Fusion polypeptides with enhanced pharmacokinetic properties

FIELD: medicine, polypeptides.

SUBSTANCE: invention relates to fusion polypeptides with enhanced pharmacokinetic properties. Fusion polypeptides comprising enhancing peptide sequences associated with the core polypeptide possess with the enhanced pharmacokinetic properties, such as prolonged half-time period. Also, invention relates to methods for enhancing pharmacokinetic properties of any core polypeptide by binding the enhancer peptide sequences with the core polypeptide. Proposed core polypeptides can comprise any pharmacologically useful peptide that can be used, for example, the therapeutic or prophylactic agent. The advantage of invention involves the enhancing of pharmacokinetic properties of polypeptides.

EFFECT: enhanced pharmacokinetic properties of polypeptides.

52 cl, 18 dwg, 14 tbl, 11 ex

 

This application is a partial continuation of application serial No. 09/350641 filed July 9, 1999, which is a partial continuation of application serial No. 09/315304, filed may 20, 1999, which is a partial continuation of application serial No. 09/082279, filed may 20, 1998, the full content of which is incorporated into this description by reference.

1. Introduction

The present invention relates to an enhancer peptide sequences, originally derived from protein sequences shell retrovirus (Dr)that enhance the pharmacokinetic properties of any crustal polypeptide with which they are associated. Partially invention is based on the discovery of the fact that the hybrid polypeptides, including enhancer peptide sequence associated with crustal polypeptide, have enhanced pharmacokinetic properties, such as increased half-life. The invention also relates to new antimutagennym and/or antiviral peptides, including peptides, which contain the enhancer peptide sequence, and to methods of using such peptides. The invention also relates to methods of enhancing the pharmacokinetic properties of any crustal polypeptide by binding enhancer peptide sequence with aravim the polypeptide. Used in the practice of implementation of the present invention crust polypeptides can include any pharmaceutically useful peptides that can be used, for example, as a therapeutic or prophylactic agent. In a non-limiting embodiment, the present invention is illustrated using an example that shows that the hybrid polypeptide, including, for example, a core polypeptide of HIV associated with the enhancer peptide sequences, is a powerful, non-toxic inhibitor of HIV-1, HIV-2 and SIV (SIV) infection. In addition, the enhancer peptide sequence according to the present invention associated with crustal polypeptide respiratory syncytial virus (RSV) and crustal polypeptide receptor, luteinizing hormone (LH-WG). In each case it is shown that the hybrid polypeptide has enhanced pharmacokinetic properties, and hybrid polypeptide of RSV exerts significant anti-RSV activity.

2. Background of invention

Polypeptide products have a wide range of applications as a therapeutic and/or prophylactic agents for the prevention and treatment of disease. Many polypeptides are capable of handling the biochemical or physiological processes with the intention of either prevention or relief related the data with disease symptoms. For example, polypeptides such as viral or bacterial polypeptides, successfully used as vaccines for the prevention of pathological conditions. In addition, peptides successfully used as therapeutic agents for the treatment of disease. Such peptides fall into different categories, such as, for example, hormones, enzymes, immunomodulators, whey proteins and cytokines.

Polypeptides, in order to provide the desired biological or therapeutic effect on the target areas, must be present in appropriate concentrations in areas of application. In addition, should mainly be supported by their structural integrity. On this basis, indications for use of the compositions of polypeptides as therapeutic agents are determined by the chemical nature and characteristics of polypeptides, such as their size and complexity, the conformational requirements, stability, support which is often difficult, and the solubility profiles. Pharmacokinetic properties of any specific therapeutic peptide dependent on the biological availability of the specified peptide, the nature of its distribution in the body and subsequent excretion from the body.

Since many biologically active substances, such as peptides and proteins are rapidly destroyed in the body, is very important to develop an effective system to maintain a stable concentration of peptide in the blood line, to increase the efficiency of action of these peptides and to minimize the occurrence of side effects and their severity.

3.1. A brief description of the invention

The present invention relates, first, to the enhancer peptide sequences, originally derived from protein sequences membranes (Dr) of various retroviruses, for example from HIV-1, HIV-2 and SIV that enhance the pharmacokinetic properties of any crustal polypeptide with which they are associated. The basis of the invention lies in the discovery of the fact that, when disclosed enhancer peptide sequence associated with any crustal polypeptide, the resulting hybrid polypeptide has enhanced pharmacokinetic properties, including, for example, increased half-life and reduced clearance compared to only one crustal polypeptide. The present invention also relates to such hybrid polypeptides and crustal polypeptides and to new peptides which exhibit antipathogen activity, antiviral activity and/or the ability to modulate intracellular processes involving collapsed helical peptide structure. These peptides include peptides that contain enhancer peptide sequence.

The crust polypeptides may include the substance of any of the peptides, which can be introduced into a living system, such as any of the peptides can function as a therapeutic, prophylactic or imaging tools that are useful for the treatment or prevention of disease or for use in diagnostic or prognostic methods, including imaging techniques in vivo. Such peptides include, for example, growth factors, hormones, cytokines, angiogenic growth factors, extracellular matrix polypeptides, ligands, receptors, agonists, antagonists or inverse agonists, peptide funds target, such as imaging means, or cytotoxic funds target, or polypeptides which exhibit antipathogen and/or antiviral activity, and peptides or polypeptides that function as antigens or immunogens, including, for example, viral and bacterial polypeptides.

The invention also relates to methods of enhancing the pharmacokinetic properties of any crustal polypeptide by binding crustal polypeptide with the enhancer peptide sequences with the formation of the hybrid polypeptides.

The invention further relates to methods of application disclosed therein peptides, including hybrid polypeptides containing the enhancer peptide sequence. For example, the methods of the present izopet the deposits include ways to reduce or suppress viral infection, for example infections caused by HIV-1, HIV-2, RSV, measles virus, influenza, parainfluenza, Epstein-Barr and hepatitis, and/or processes merge cells, caused by a virus. Enhancer peptide sequence according to the present invention can additionally be used to improve in vitro or ex-vivo half-life of the crustal polypeptide, joined enhancer peptide sequences, for example enhancer peptide sequences can increase the half-life of attached core polypeptides in cell culture or sample of cells or tissue.

The invention is illustrated by examples, which show that the hybrid polypeptides containing a core polypeptide of HIV associated with the enhancer peptide sequences, exhibit significantly enhanced pharmacokinetic properties and act as a powerful nezatocheny inhibitors of HIV-1, HIV-2 and SIV infection. The invention is further illustrated by the examples in which it is shown that the hybrid polypeptides containing a core RSV polypeptide or polypeptide of luteinizing hormone, show significantly enhanced pharmacokinetic properties. In addition, the hybrid polypeptide with RSV possesses significant activity against RSV.

3.2. Definition

In the context of the present description, the peptides, polypeptid the s and proteins are defined as organic compounds, comprising two or more amino acids, covalently linked, for example, by using a peptide amide bonds. Peptides, polypeptides and proteins can also include non-natural amino acids in any modification, as well as additional amino groups and carboxyl groups, as indicated in the description. The terms "peptide", "polypeptide" and "protein" are used interchangeably when used in the present description.

Defined in the present description the peptide sequences are represented using single-letter symbols corresponding to specific amino acid residues, as shown below:

A (alanine)

R (arginine)

N (asparagine)

D (aspartic acid)

C (cysteine)

Q (glutamine)

E (glutamic acid)

G (glycine)

H (histidine)

I (isoleucine)

L (leucine)

K (lysine)

M (methionine)

F (phenylalanine)

P (Proline)

S (serine)

T (threonine)

W (tryptophan)

Y (tyrosine)

V (valine)

X (any amino acid)

The term "enhancer peptide sequence in the context of the present description means a peptide having the following consensus amino acid sequence:

"WXXWXXXI", "WXXWXXX", "WXXWXX", "WXXWX", "WXXW", "WXXXWXWX", "XXXWXWX", "XXWXWX", "XWXWX", "WXWX", "WXXXWXW", "WXXXWX", "WXXXW", "IXXXWXXW", "XXXWXXW", "XXWXXW", "XWXXW", "XWXWXXXW", "XWXWXXX", "XWXWXX", "XWXWX", "XWXW", "WXWXXXW" or "XWXXXW", where X can be any amino acid, W means tryptopha and I is isoleucine. As discussed further, the enhancer peptide sequence according to the present invention also include peptide sequence, which is broadly the same as the consensus amino acid sequence but contain amino acid substitutions, insertions or deletions, which, however, do not eliminate the ability of the peptide to enhance the pharmacokinetic properties of the crustal peptide with which it is associated, in comparison with the pharmacokinetic properties of one crustal polypeptide.

The term "core polypeptide in the context of the present description refers to any polypeptide that can be introduced into a living system, and thus represent a biologically active molecule, for example, any polypeptide that can act as pharmacologically useful peptide in the treatment or prevention of disease.

The term "hybrid polypeptide" in the context of the present description refers to any polypeptide comprising amino, carboxy or amino and carboxy terminal enhancer peptide sequence and a core polypeptide. In a typical case, the enhancer peptide sequence directly related to crustal polypeptide. It should be understood that the enhancer peptide can also join the intermediate amino acid sequence having the I between the enhancer peptide sequence and crustal peptide.

The terms "antipathogenic" and "preventing the fusion of membranes in the context of the present description relate to the ability of the peptide to inhibit or reduce the effects of the merger of two or more structures, such as cell membranes or viral membranes or flagella, in comparison with the level of fusion of the membranes, which is present between the structures in the absence of peptide.

The term "antiviral" in the context of the present description refers to the ability of the peptide to inhibit viral infection of cells flowing through, for example, merge cells, or free virus infection. This infection can be caused by the fusion of membranes that occurs in the case of viruses, coated, or other mergers involving viral structure and cell structure, for example the fusion of viral flagella and bacterial membrane during bacterial conjugation.

4. Brief description of drawings

Figure 1. Hybrid polypeptides. Enhancer peptide sequence derived from the estimated N-terminal and C-terminal interacting plots shown associated with major crustal polypeptide. Conservative enhancer peptide sequences are shaded. It should be noted that these enhancer peptide sequences can be used with either the AK N-terminal, C-terminal or N - and C-terminal additions. In addition, the enhancer peptide sequence can be added to korovou the polypeptide in a direct or reverse orientation, individually or in any possible combinations, to enhance the pharmacokinetic properties of the peptide.

Figa. Enhancer peptide sequences derived from different protein sequences membranes (Dr), reflecting the N-terminal interactive plot observed in all published to date data about the selected sequences of HIV-1, HIV-2 and SIV. End sequence "WXXWXXXI" means a consensus sequence.

Figv. Options enhancer peptide sequences derived from different protein sequences membranes (Dr), reflecting the C-terminal interacting plot observed in all published to date data about the selected sequences of HIV-1, HIV-2 and SIV. End sequence "WXXXWXWX" means a consensus sequence.

Figure 3. Comparison of titers of HIV-1 in tissues of mice SCID-huPBMC infected with HIV-1 9320, according to the P24 levels obtained in tests on culture huPBMC. Figure 3 shows a comparison of the levels of inhibition of virus T20 and T in vivo.

Figa-4V. The pharmacokinetic profile T in plasma in comparison with the level of control is with crustal option T in rats CD after/in the injection site within 2 hours (figa) and 8 hours (pigv). Polypeptide T is a core polypeptide and T is a core polypeptide that is associated with the enhancer peptide sequences.

Figure 5. The pharmacokinetic profile T in plasma in comparison with the control variant T20 rats CD after/in the injection site. Polypeptide T is a hybrid polypeptide formed from crustal polypeptide (T)associated with the enhancer peptide sequences. T20: n=4; T: n=3.

6. Comparison of activity and cytotoxicity T20/T against HIV-1/IIIb.

7. Direct linking T with the design based on Dr - M41Δ178.125I-T1249 cleaned to the maximum achievable specific activity by HPLC. Shows saturation at the binding M41Δ178 (protein merge from ectodomain Dr not containing the amino acid sequence T20)immobilized on the microtiter plate in a concentration of 0.5 mg/ml

Fig. The process of Association/dissociation T over time. The results show that125I-T1249 and125I-T20 have similar values of the affinity of binding of 1-2 nm. The initial rate of Association and dissociation for125I-T1249 significantly lower than for the125I-T20. The dissociation of the bound ligand with a radioactive label is measured by adding its peptide to a final concentration of 10 MK is in 1/10 of the total environment for testing.

Fig.9. Competition for binding T with M41Δ178. Its T and T20 titrated in the presence of the same concentrations125I-T1249 or125I-T20. The ligand is added immediately after the start of incubation, its peptide.

Figa-10V. The pharmacokinetic profile of the hybrid polypeptide of RSV - T (10A) and T (10B) in the blood plasma of rats CD in comparison with T.

Figa. Test for reducing belascoaran. Hybrid polypeptide T able to inhibit infection with RSV value IR50equal to 2.6 mg/ml

Figv. Test for reducing belascoaran shows that the hybrid polypeptide of RSV T, T and T inhibit RSV infection.

Figa and 12V. The pharmacokinetic profile of the hybrid polypeptide of luteinizing hormone - T in comparison with T in the blood plasma of male rats CD. Polypeptide T is a core polypeptide of luteinizing hormone, polypeptide C is a hybrid polypeptide comprising a core polypeptide that is associated with the enhancer peptide sequences.

Fig. Hybrid polypeptide sequence derived from different crustal polypeptides. Core polypeptide sequence shown in shaded form. Unshaded sequence at the amino - and carboxy-ends point to the enhancer peptide sequence.

Figa-Century Spectra krugovorota (KD) for T in solution (phosphate buffer solution, pH 7) one (10 μm at 1°, figa) and in combination with a peptide comprising 45 residues from Dr HR1 binding domain (T); black squaremeans calculated CD spectrum predicted by the model of "lack of cooperation", while the actual CD spectra indicated the blackened circle

Fig. The results of polyacrylamide gel electrophoresis, showing protection by T design based Dr - M41Δ178 from cleavage by proteinase K; lane 1: the primer marker; lane 2: untreated M41Δ178; lane 3: M41Δ178, incubated with proteinase K; lane 4: untreated T; track 5: T, incubated with proteinase K; lane 6: M41Δ178, incubated with T; track 7: incubation T and M41Δ178 before adding proteinase K.

Figa-S. Pharmacokinetics T in albino rats sprag-Share (Sprague-Dawley); figa: pharmacokinetics T in the case of the introduction of a single dose by continuous subcutaneous infusion; figv: pharmacokinetics C in plasma with the introduction of its subcutaneous injection (p/C) or intravenous injection (in/in); figs: kinetic analysis T in the lymph and blood plasma after intravenous injection.

Figa-Century Pharmacokinetics T in cynomolgus monkeys;

figa: pharmacokinetics in plasma of a single dose is 1249 in 0.8 mg/kg, injected subcutaneous (s/C), intravenous (IV) or intramuscular (I/m) injection; figv: pharmacokinetics in plasma T after subcutaneous administration of three different doses (0.4 mg/kg, 0.8 mg/kg and 1.6 mg/kg).

Figa-18D. Antiviral activity exhibited by peptides DP397T (--○--) and T (--▵--) in different resistant T strains of HIV-1, according to test infectivity method Magi-CCR-5; solid (top) and dashed (lower) horizontal line on each drawing indicate the levels of 50% and 90% reduction of infectivity of HIV-1, respectively; figa: antiviral activity shown DP397, T and T in HIV-1 strain RF-649; figv: antiviral activity shown DP397, and T T in HIV-1 strain DH012-649; figs: antiviral activity shown DP397, T and T in HIV-1 strain 3'ETVQQQ; Fig 18D: antiviral activity shown DP397, T and T in HIV-1 strain SIM 649.

5. Detailed description of the invention

In the present description of the disclosed peptide sequences, called enhancer peptide sequences derived from different protein sequences shell retrovirus (Dr), which can enhance the pharmacokinetic properties of crustal polypeptides to which they are attached. Such enhancer peptide sequences can be used in the methods of the Ah, aimed at enhancing the pharmacokinetic properties of any crustal polypeptide by binding enhancer peptide sequences with crustal polypeptide with the formation of the hybrid polypeptide with enhanced pharmacokinetic properties compared with one crustal polypeptide. The half-life of the crustal polypeptide, to which are attached one or more enhancer peptide sequences can also be increased in vitro. So, for example, attached enhancer peptide sequences can increase the half-life of the polypeptide in the crustal case in cell culture, tissue culture or in a sample obtained from the patient, such as a cell, tissue or other samples.

Core polypeptides in the composition of the hybrid polypeptides according to the present invention can include any peptide, which can be introduced into a living system, for example, any peptide that can act as therapeutic or prophylactic agent, useful for the treatment or prevention of disease, or as a visualizing agent, useful for the visualization of structures in vivo.

In the description of the peptides, including peptides, which contain the enhancer peptide sequence, showing antipathogen and/or antiviral activity. In addition to t the th, describes the ways in which such peptides, including the methods used to reduce or suppress viral infection and/or merge cells, caused by a viral infection.

5.1. Hybrid polypeptides

Hybrid polypeptide according to the present invention include at least one enhancer peptide sequence and a core polypeptide. Preferably, the hybrid polypeptides according to the present invention include at least two enhancer peptide sequence and a core polypeptide, and at least one enhancer peptide located at the amino-end of the crustal polypeptide and at least one enhancer peptide sequence present in a hybrid polypeptide that is at the carboxy-end of the crustal polypeptide.

Enhancer peptide sequence according to the present invention include peptide sequence, originally derived from different protein sequences shell retrovirus (Dr), including sequences of HIV-1, HIV-2 and SIV, and made them specific variations or modifications described below. A core polypeptide can include any peptide sequence, preferably any peptide sequence that can be introduced into a living system, including, for example, peptides, dedicated to superior quality products is obtained for purposes of treatment, prevention or rendering.

In a typical case, the length of the hybrid polypeptide ranges from about 10 to about 500 amino acid residues, with a preferred length is from about 10 to about 100 amino acid residues, and most preferred is a length from about 10 to about 40 amino acid residues.

Without limiting the discussion to any particular theory, it should nevertheless be noted that the structure of the protein shell is such that the alleged plot α-helix located at the C-terminal site of the protein, most likely, is connected with a plot "zipper" leucine, located at the N-terminal site of the protein. Comparative analysis of the structure plots do with N-terminal and C-terminal enhancer sequences, carried out in all published to date, the work on the selection of sequences of HIV-1, HIV-2 and SIV, leads to the identification of a consensus amino acid sequence.

In particular, we have identified below the consensus amino acid sequence reflecting the consensus enhancer peptide sequence (consensus sequence listed in forward and reverse orientations, as these enhancer peptide sequence can use is to use either in a straight line, or in the reverse orientation): "WXXWXXXI", "WXXWXXX", "WXXWXX", "WXXWX", "WXXW", "WXXXWXWX", "XXXWXWX", "XXWXWX", "XWXWX", "WXWX", "WXXXWXW", "WXXXWX", "WXXXW", "IXXXWXXW", "XXXWXXW", "XXWXXW", "XWXXW", "XWXWXXXW", "XWXWXXX", "XWXWXX", "XWXWX", "XWXW", "WXWXXXW" or "XWXXXW", where X can be any amino acid, W means the tryptophan and I is isoleucine. Direct orientation of the consensus amino acid sequences shown in figures 1 and 2.

In a typical case, the enhancer peptide sequence is approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid residues in length, with preferred length is from about 4 to about 20 residues, more preferred is a length from about 4 to about 10 residues, and most preferred is a length from about 6 to about 8 residues.

In a preferred variant of the invention, the enhancer peptide sequences that can be used to enhance the pharmacokinetic properties of the resulting hybrid polypeptides include specific enhancer peptide sequence shown in figure 2, 13 and in Table 1. Among the most preferred enhancer peptide sequences include those which comprise the following amino acid sequence: "WQEWEQKI" and "WASLWEWF".

To illustrate but not to limit, below in Table 1 are presented the ENES amino acid sequence, which reflect the preferred options enhancer peptide sequences from among the enhancer peptide sequences according to the present invention. It should be understood that although shown only the direct orientation of these sequences, the reverse orientation of the given sequences is also included in the scope of the present invention. For example, if the following shows the direct orientation of the enhancer peptide sequence "WMEWDREI", and its reverse orientation, i.e. the "IERDWEMW", is also included.

Table 1
WMEWDREI (SEQ ID NO:1544)
WQEWERKV (SEQ ID NO:1545)
WQEWEQKV (SEQ ID NO:1546)
MTWMEWDREI (SEQ ID NO:1547)
NNMTWMEWDREI (SEQ ID NO:1548)
WQEWEQKVRYLEANI (SEQ ID NO:1549)
NNMTWQEWEZKVRYLEANI (SEQ ID NO:1550)
WNWFI (SEQ ID NO:1551)
WQEWDREISNYTSLI (SEQ ID NO:1552)
WQEWEREISAYTSLI (SEQ ID NO:1553)
WQEWDREI (SEQ ID NO:1554)
WQEWEI (SEQ ID NO:1555)
WNWF (SEQ ID NO:1556)
WQEW (SEQ ID NO:1557)
WQAW (SEQ ID NO:1558)
WQEWEQKI (SEQ ID NO:1559)
WASLWNWF (SEQ ID NO:1560)
WASLFNFF (SEQ ID NO:1561)
WDVFTNWL (SEQ ID NO:1562)
WASLWEWF (SEQ ID NO:1563)
EWASLWEWF (SEQ ID NO:1564)
WEWF(SEQ ID NO:1565)
EWEWF (SEQ ID NO:1566)
IEWEWF (SEQ ID NO:1567)
IEWEW (SEQ ID NO:1568)
EWEW (SEQ ID NO:1569)
WASLWEWF (SEQ ID NO:1570)
WAGLWEWF (SEQ ID NO:1571)
AKWASLWEWF (SEQ ID NO:1572)
AEWASLWEWF (SEQ ID NO:1573)
WASLWAWF (SEQ ID NO:1574)
AEWASLWAWF (SEQ ID NO:1575)
AKWASLWAWF (SEQ ID NO:1576)
WAGLWAWF (SEQ ID NO:1577)
AEWAGLWAWF (SEQ ID NO:1578)
WASLWAW (SEQ ID NO:1579)
AEWASLWAW (SEQ ID NO:1580)
WAGLWAW (SEQ ID NO:1581)
AEWAGLWAW (SEQ ID NO:1582)
DKWEWF (SEQ ID NO:1583)
IEWASLWEWF (SEQ ID NO:1584)
IKWASLWEWF (SEQ ID NO:1585)
DEWEWF (SEQ ID NO:1586)
GGWASLWNWF (SEQ ID NO:1587)
GGWNWF (SEQ ID NO:1588)

In another preferred embodiment, specific enhancer peptide sequence according to the present invention include an enhancer peptide sequence shown in figure 2, 13 and in Table 1 are conservative substitutions of amino acids in one, two or three positions, and these amino acid substitution does not eliminate the ability of the enhancer peptide sequences to enhance the pharmacokinetic properties of hybrid polypeptide in comparison with the corresponding member crustal polypeptide.

Most preferably, such substitutions result in an enhancer peptide sequences that fall within the scope of the consensus sequences in the enhancer peptide sequence. In these cases, substitution mainly occur at amino acid residues corresponding to position "X"indicated in the above consensus amino acid sequences in figure 1 and 2. The term "conservative substitution" refers to substitution when the specified substitution is of amino acid residues with similar values of the charge, size and/or hydrophobicity/hydrophilicity, Thu and the substituted amino acid. Marked characteristics of the amino acids is well known to specialists in this field of technology.

The present invention also relates to the enhancer peptide sequences comprising the amino acid sequence shown in figures 1, 2, 13 and in Table 1, which are all identical, with the exception that these enhancer peptide sequences include one or more amino acid supplements (generally not more than about 15 amino acid residues in length), deletions (e.g., truncation at the amino group or on end parts) or non-conservative substitutions, which however does not eliminate the ability of the obtained enhancer peptide sequences to enhance the pharmacokinetic properties of crustal polypeptides to which they are attached, in comparison only with the core polypeptides without enhancer peptide sequences.

Supplements are mostly not longer than 15 amino acid residues, and may include additives, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive amino acid residues. Preferably the total number of amino acid residues added to the original enhancer peptide, does not exceed about 15 amino acid residues, more preferably is not more than about ten amino acid residues, and most the e is preferably not more than about five amino acid residues.

Deletions preferably represent such deletions, which include not more than about 3 amino acid residue (either consistent or inconsistent balance), more preferably deletions, including 2 amino acid residue and is most preferably a deletion of one amino acid residue. Mainly deletions represent amino acid residues corresponding to residues "X" in the consensus sequences of the enhancer peptide.

Enhancer peptide sequence according to the present invention also include specific enhancer peptide sequence shown in figure 2, 13 and in Table 1, containing one, two or three non-conservative amino acid substitution, preferably two such substitutions and most preferably, the presence of one such substitution. The term "non-conservative" in relation to the substitution refers to the substitution of amino acid residues with dissimilar indices of charge, weight, and/or hydrophobicity/hydrophilicity compared to the replaced amino acid residue. Such characteristics of the amino acids is well known to specialists in this field of technology.

In addition, it is not necessary that the amino acid substitution was restricted to the genetically encoded amino acids that applies equally is to certain preferred variants of the invention. In fact, the peptides may contain amino acids that are not genetically encoded. So, in addition to natural genetically encoded amino acids of amino acid residues in the peptides may be replaced natural non-coding amino acids and synthetic amino acids. Such substitution can also take place inside the core polypeptides in a hybrid polypeptides according to the present invention, regardless of whether they enhancer sequence/sequence-specific hybrid polypeptide.

Among the common amino acids, which allow useful substitutions include, but are not limited to the above list, the following: β-alanine (β-Ala) and other omega-amino acids such as 3-aminopropionic acid, 2,3-diaminopropionic acid (Dpr), 4-aminobutyric acid, etc.; α-aminoadamantane acid (Aib); ε-aminohexanoic acid (Aha); δ-aminosalicilova acid (Ava); N-methylglycine or sarcosine (MeGly); ornithine (Orn); citrulline (Cit); t-butylene (t-BuA), t-butylglycol (t-BuG), N-methylisoleucine (Melle); phenylglycine (Phg); cyclohexylamine (Cha); norleucine (Nle); nafcillin (Nal); 4-chlorophenylalanine (Phe(4-Cl)); 2-forfinally (Phe(2-F)); 3-forfinally (Phe(3-F)); 4-forfinally (Phe(4-F)); penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic); #x003B2; -2-titillans (Thi); methanesulfonic (MSO); homoarginine (hArg); N-acetylized (AcLys); 2, 4-diaminobutane acid (Dbu); 2,3-diaminobutane acid (Dab); p-aminophenylalanine (Phe(pNH2)); N-methylvaline (MeVal); homocysteine (hCys); homophenylalanine (hPhe), homoserine (hSer); hydroxyproline (Hyp); gemopolis (hPro); -methylated amino acids, analogues of cyclic amino acids (used, for example, to limit the structure of the amino acid residues to specific conformational States, for example, αα' ββ'-substituted cyclic amino acids, such as 1-aminocyclopentane-carboxylic acid (cycloleucine) and ββ-cyclopentadien-β-mercaptopropionic acid (see, e.g., Hruby et al., 1990, Biochem. J. 268: 249-262) and peptide or Oligopeptide (N-substituted amino acids, such as N-substituted glycine; see, for example, Simon et al., 1972, Proc. Natl. Acad. Sci. USA 89: 9367-9371).

Despite the fact that in most cases, the amino acids in the peptide are replaced by L-enantiomeric amino acids, the substitutions are not limited to L-enantiomeric amino acids. So, the definition of "mutated" or "modified" forms against amino acids also covers those situations in which the L-amino acid is replaced with an identical D-amino acid (for example, L-Arg - D-Arg or D-amino acid of the same category or subcategory (e.g., L-Arg - D-Lys), and Vice versa. the hat substitution can also take place on crustal polypeptides in a hybrid polypeptides according to the present invention, regardless of whether they enhancer sequence/enhancer sequence-specific hybrid polypeptide.

In addition to the above-described amino acid substitutions substitution in the side groups can be done with the introduction, for example, a methyl group or pseudotolerance groups with different electronic properties (see, e.g., Hruby et al., 1990, Biochem. J. 268: 249-262). In addition, can be introduced double bond between adjacent carbon atoms in amino acids and cyclic peptides or can be formed analogues with the introduction of covalent bonds, such as in the case of formation of amide bond between a - and C-end, between two side chains or between a side chain and - or-end of the peptide. Such substitution can also take place inside the core polypeptides in a hybrid polypeptides according to the present invention, regardless of whether they are or not in the enhancer sequence/enhancer sequence-specific hybrid polypeptide.

The core and hybrid polypeptides according to the present invention may also be conjugated with one or more chemical groups. Chemical group used for conjugation, preferably not possess significant toxicity or immunogenicity, i.e. any toxicity of the sludge is immunogenicity, observed for the conjugate crustal or hybrid polypeptide is negligible (i.e. less than 50%) is higher than any toxicity or immunogenicity, celebrated for unmodified crustal or hybrid polypeptide. Chemical modification of the crustal and/or hybrid polypeptides are aimed at influencing the pharmacokinetic properties of the polypeptide. These effects include reducing or increasing the efficiency of medicines, stability, bioavailability, clearance, immunogenicity and the half-life in vivo, and the effect on the catabolism of the polypeptide, its directional migration and localization.

In one embodiment of the invention teach the conjugation of the hybrid polypeptides with one or more chemical groups, carrying it like a cow and enhancer parts of the polypeptide. In another embodiment of the invention, such modifications can be carried out either on a cow parts of the polypeptide or part of the enhancer peptide hybrid polypeptides. In yet another embodiment, the invention is modified only crustal part of the polypeptide in a hybrid polypeptide. In yet another embodiment of the invention a core polypeptide modify one or more chemical groups, and such a core polypeptide is not predstavljaet a part of the hybrid polypeptide. For example, a core polypeptide, such as T (Ac-TALLEQAQIQQEKNEYELQKLDK-NH2can be modified using one or more chemical groups.

Examples of chemical groups that are useful for the implementation of the conjugation include polymers of non-protein nature, such as polyols. Other chemical groups include proteins, such as albumin or immunoglobulin, as well as carbohydrates, such as those carbohydrates that occur naturally in glycoproteins. For modification of proteins also use acid, DL-amino acid and polyvinylpyrrolidone. An overview of the polymer-modified peptides is given in the work Burnham (Burnham, Am. J. Hosp. Pharm. 51: 210-18 (1994)), which is fully described in the present description by reference.

Polyol, for example, can be anywhereman with crustal or hybrid polypeptide by one or more amino acid residues, including, for example, residues of lysine, cysteine and histidine. Used polyol may be any water-soluble poly(accelerometry) polymer and may contain linear or branched chain. Suitable polyols include polyols, substituted by one or more hydroxyl provisions of the chemical group such as an alkyl group containing from one to four carbon atoms. In a typical case, the polyol is a floor is(allenglish), such as poly(ethylene glycol) (PEG) and therefore, for ease of discussion, the remainder of the description will refer to illustrative variant of the invention, in which the polyol used is a PEG and the way conjugation polyol with crustal or hybrid polypeptide is called "tahilramani". However, specialists in the art it is known that can be used and other polyols, such as, for example, poly(propylene glycol), and copolymers of polyethylene-polypropyleneglycol, using the techniques of conjugation specified in the present description with reference to PEG. Overview on methods for the modification of bioactive molecules is given in the work of Inada et al. (Inada et al., Trends Biotechnol. 13: 86-91 (1995)), which is fully described in the present description by reference.

The degree of tahilramani crustal or hybrid polypeptide can be adjusted to increase to the desired level, the half-life in vivo in comparison with the corresponding non-pegylated protein. The half-life of pegylated crustal or hybrid polypeptide may be increased gradually increasing the degree of tahilramani. It is shown that the modification of proteins with the use of PEG or PEG derivatives can increase to the desired level of efficacy of medicinal product, its biological act shall want to make, stability (including heat resistance, chemical denaturing factors and proteolysis), adsorption/absorption and half-life in vivo, and reduces to the desired level, the rate of clearance and immunogenicity (U.S. patent No. 6025325; Westerman et al., Int. J. Cancer 76: 842-50 (1998); Conover et al., Artif. Organs 21: 907-15 (1997); Tsutsumi et al., J. Pharmacol. Exp. Ther. 278: 1006-11 (1996); Kaneda et al., Invasion Metastasis 15: 156-62 (1995); Inada et al., Trends Biotechnol. 13: 86-91 (1995); Paige et al., Pharm. Res. 12:1883-88 (1995); Satake-Ishikawa et al., Cell Struct. Funct. 17: 157-60 (1992); Tanaka et al., Cancer Res. 51: 3710-3714 (1991), is included in its entirety in the present description as a reference).

The average molecular weight of the PEG can vary from approximately 500 to approximately 30,000 daltons (D), preferably from about 1000 to about 25,000, and more preferably from about 4000 to about 20,000 D. In one embodiment, the invention tahilramani carried out using PEG with molecular weight of approximately 5000 D (hereinafter referred to as "PEG(5000)"). In another embodiment of the invention using PEG branched chain, containing two chains of about 10,000 On each.

Commercially available preparations of PEG, which is acceptable for use within the present invention relate to inhomogeneous drugs sold in accordance with their molecular weight. For example, preparations of PEG(5000) typically contain molecules of varying n is considerably molecular weight, usually within +/-500 D. Describes numerous methods tahilramani proteins. See, for example, U.S. patent No. 4179337, is included in its entirety in the present description by reference, which discloses the conjugation of a large number of hormones and enzymes with PEG and polypropylenglycol with the formation of the physiologically active non-immunogenic compositions.

Reaction conditions the binding of PEG to protein vary depending on the nature of the target protein, the desired degree of tahilramani, the type of PEG or its derivative and localization of the target point. Other factors include stability, reaktsionnosposobnykh and antigenicity connection with PEG. Usually the reaction of PEG with at least one terminal hydroxyl group, with the binder with the formation of the activated PEG-carrying terminal reactive group. Then spend the reaction specified reactive group with αand ε-amines of proteins with the formation of covalent bonds. Amine groups can konjugierte with the hydroxy-group of the PEG with the formation of amide linkages. Carboxyl groups can konjugierte with amine groups crustal or hybrid polypeptide with the formation of the amide bond and a hydroxy-group of the PEG with the formation of ester. While the other end of the PEG molecules may be "blocked" preaction sposobnoi chemical group, such as a methoxy group, with the formation of, for example, alkyl PEG, such as methoxy-PEG (MPEG), which reduces the level of education cross-stitched PEG complexes with protein molecules.

In the technique known several types of linker groups used for conjugation crustal, enhancer or hybrid polypeptide with PEG. Examples of linker groups described, for example, in U.S. patent No. 4609546, in U.S. patent No. 4847325, in U.S. patent No. 4902502, in U.S. patent No. 5034514 and in U.S. patent No. 5122614. In one embodiment of the invention, the linker use the enhancer peptide sequence.

Suitable activated PEG can be obtained as a result of a number of relevant reactions. So, for example, N-hydroxysuccinimidyl ester of PEG (M-NHS-PEG) can be obtained from PEG-nanometrology ether (commercially available from Union Carbide) when carrying out the reaction of N,N'-dicyclohexylcarbodiimide (DCC) with N-hydroxysuccinimide (NHS) in accordance with the method Buckmann and Merra (Buckmann and Merr, Makromol. Chem., 182: 1379-1384 (1981)).

In addition, end the hydroxy-group of the PEG can be converted into the amino group, for example, when interacting with tierbroker with the formation of PEG-Br and when conducting subsequent aminolysis with an excess of ammonia with the formation of PEG-NH2. This is followed by the conjugation of PEG-NH2with interesting the m protein using standard bonding agents, such as the reagent To Woodward. Next end group-CH2HE PEG can be transformed into an aldehyde group, for example, oxidation with MnO2. Then spend the conjugation of the aldehyde group with the protein in the reaction reductive alkylation using a reagent such as Lamborghini. Amino acids can also be linked to PEG their amino groups through a suitable linkage, such as, for example, urethane group. Unnatural amino acid norleucine with the purpose of binding to amino groups of the protein can be activated by its carboxyl group to Succinimidyl of ester (Zalipsky et al., Int. J. Peptide Protein Res. 30:740 (1987); Sartore et al., Appl. Biochem. Biotech. 27: 45 (1991)). A General overview of tahilramani described in Zalipsky and Lee in "Poly (Ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications", J.M.Harris, Ed., Plenum, NY, Chap 21 (1992), which is included in its entirety in the present description by reference.

Alternative suitable for application in the framework of the present invention, the activated PEG include, but are not limited to the above list, the following: acrylate-PEG -, aldehyde-PEG -, allyl-PEG, amino-PEG, amino-PEG, esters of amino-PEG, ω-amino α-carboxyl-PEG, benzo-triazolinones, Biotin-PEG, t-BOC-PEG, carbonyldiimidazole-PEG, karboksimetilirovaniya PEG, epoxide-PEG, Fmoc-PEG, fluorescein-PEG -, hydrazide-PEG ω -hydroxy α-amino-PEG, ω-hydroxy α-carboxyl-PEG, isocyanate-PEG, maleimide-PEG, methacrylate ester PEG-NHS-maleimide, NHS-vinylsulfonic, p-nitrophenylarsonic-PEG, orthopaedically, PEG, phospholipid-PEG, propionic acid-PEG, the silane-PEG, succinate-PEG, succinimidylester-PEG (SBA-PEG), succini-meilowy ester of amino-PEG, Succinimidyl ester carboxymethylamino PEG, Succinimidyl-propionate-PEG (SPA-PEG-SPA-PEG), Succinimidyl-PEG - (SS-PEG-SS-PEG), thiol-PEG, vinylsulfonic-PEG, which can be purchased from various suppliers. So, for example, Shearwater Polymers, Inc. (Huntsville, Ala.) sells M-NHS-PEG as "SCM-PEG", and succinimidylester MPEG ("SC-PEG") and succinimidylester MPEG (SPA-PEG").

Specific amino acids, including hybrid or a core polypeptide, may be modified, for example, for the purposes of preventing and/or facilitate tahilramani certain amino acid residues. In one embodiment of the invention potential areas tahilramani can be inactivated by modifying those amino acid residues that can be tahilramani. For example, all amino acid residues having a free amino group in the crust or hybrid polypeptide can be protected, which prevents tahilramani specified by the residues. Suitable protective groups include, but are not limited to the above list, the following: tert-butoxycarbonyl (t-Side) and N-9-fluorenylmethoxycarbonyl (Fmoc). May be any protective group for amino group. Several such protective groups described in (Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1981, pp.323-334; and Fields and Noble, Int. J. Pept. Protein Res. 35: 161-214 (1990), included in the present description fully as references. If desired, the protective group can be subsequently removed using routine chemical procedures, such as treatment with piperidine in dimethylformamide for Fmoc or when processing triperoxonane acid for t-Side.

Alternative amino acid residues crustal or hybrid polypeptide sensitive tahilramani, can be substituted for amino acid residues that are resistant to tahilramani, for example, by site-directed mutagenesis. In addition, the hybrid polypeptides can be used enhancer peptide sequences that do not contain amino acid residues that are sensitive to tahilramani.

In another embodiment of the invention, one or more amino acid residues of the crustal or hybrid polypeptide can be modified in order to enter additional areas tahilramani. So, voltage is emer, one or more amino acid residues that are sensitive to tahilramani, can be used to replace or added to the polypeptide using any method known in the art, such as standard methods of synthesis or recombinant methods by site-directed mutagenesis (Zoller et al., Nucl. Acids Res. 10: 6487 (1987); Carter et al., Nucl. Acids Res. 13: 4331 (1986)).

Tahilramani crustal or hybrid polypeptide may also be undertaken in order to influence the localization of the site of tahilramani or on the degree of tahilramani. Tahilramani only part of the possible areas tahilramani in the crust or hybrid polypeptide can be performed using any method known in the art. For example, the degree of tahilramani can be controlled by reaction conditions such as the change of the coefficient of molar ratio of the polypeptide to the PEG, the duration of the reaction or the temperature at which the reaction is carried out. After purification of the pegylated polypeptide, for example, by using ion-exchange chromatography, the degree of tahilramani polypeptide can be determined, for example, the analysis in the LTO-PAG. Pegylated protein can be stored in conditions that use any known in the art environment for storage, including storage at -20°With the FBI buffet is e (pH of 7.3).

These approaches can be combined in order to regulate the number and localization of sites of tahilramani along the crustal or hybrid polypeptide. Tahilramani specific amino acid residue may be made when combining methods of chemical protection and reactions tahilramani in the synthesis of crustal, enhancer or hybrid polypeptides. The use of different protective groups in protecting and deprotecting at different points during peptide synthesis allows to reach any(s) of amino acid(s) residue(s) to implement tahilramani. Thus, this technique can be used for selective modification of any chemical group specific amino acid residue or any part thereof in the crust, enhancemen or hybrid polypeptide. For example, it is possible to carry out chemical protection partially synthesized oligopeptides using the first protective groups to prevent tahilramani amino acid residues, which could be otherwise sensitive tahilramani.

Further synthesis of the polypeptide can be completed using the second protective group, which will also protect amino acid residues, which otherwise would have been sensitive to tahilramani. The second protective group must have the ü more mobile, than the first, so that at the completion of the synthesis of specified movable protective group could be removed, allowing tahilramani only now unprotected interested amino acid residues (Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1981).

In one non-limiting example, may be selectively modified by any one amino acid residue of T (AU-TALLEQAQIQQEKNEYELQKLDK-NH2), which is sensitive to tahilramani (e.g., lysine residues). So, for example, can be synthesized KLDK, and ε-NH2the group may be subject to deprotection and then pagalilauan. Peptide synthesis is completed by the formation of the crustal polypeptide, which contains in pegylated form only two of the three lysine residues.

In another non-limiting example, a large part of the N-terminal lysine residues in T can be modified in the implementation of the synthesis NEYELQKLDK in the case of protection ε-NH2groups of lysine residues less mobile protective group. After completion of protein synthesis, in which ε-NH2a group of the most remote to the N-end lysine contains more movable protective group specified by N-terminal lysine is subjected to deprotection and tahilramani. Specialists in this field it is clear that strategies such as those described, can be used for selective modification and the achievement of the possible amino acid(s) residue(s) within the crustal, enhancer or hybrid polypeptide. Such protective groups and their mobility is known from the prior art, in particular in the direction of Greene (Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1981).

One or more amino acid sequences can also be conjugated with crustal or hybrid polypeptides. Used amino acid sequence can be any amino acid sequence that is characterized by a long half-life or can achieve such a merger proteins. In one embodiment of the invention used protein is of human origin. In a preferred embodiment of the invention this protein is an albumin human. However, experts in this field it is clear that other proteins, such as immunoglobulins, also can be used in conjugation method described in the description with reference to albumin. For example, a core, enhancer or hybrid polypeptide can be conjugated with a representative of a family of immunoglobulins or fragments of immunoglobulins. In one embodiment of the invention carried out the merger of the crustal, enhancer or hybrid polypeptide with Fc domain of human IgG1.

In another variationbetween of the invention the polypeptide modified by attaching modification nepataisomos nature of the polyol, and modification of amino acid sequence, such as modification with PEG and albumin.

Amino acid sequence may be attached to either the C-terminal or N-terminal parts of the crustal, enhancer or hybrid polypeptide, either in the form of side chains of the polypeptide. Amino acid sequences can be added to korovou or hybrid polypeptide or during synthesis of the polypeptide, or after its completion. An alternative can be designed which allows to obtain protein, so that the obtained recombinant amino acid sequence to attach then to korovou and/or hybrid polypeptide. For example, the albumin can be merged with any end of the crustal polypeptide. Optionally, enhancer peptide sequence can then be placed on the free end of the crustal polypeptide and/or at the end containing attached recombinant protein. In one embodiment of the invention the amino acid sequence can serve as a linker between crustal and enhancer polypeptides or may be part of it.

It is shown that the modification of proteins by fusion with the amino acid sequences, such as albumin, increases to the desired level of efficacy of medicinal product, it shall stabilnosti, biological activity, and reduces to the desired level clearance and toxicity (Kratz et al., Arch. Pharm. (Weinheim) 331: 47-53 (1998); Syed et al., Blood 89: 3243-3252 (1997); Makrides et al., J. Pharmacol. Exp. Ther. 277: 534-42 (1996); Breton et al., Eur. J. Biochem. 231: 563-69 (1995); Paige et al., Pharm. Res.12: 1883-88 (1995)).

Albumin has a high polimorfismo so many variants have been identified (Weitkamp et al., Ann. Hum. Genet. 37: 219 (1973)). The sequence of albumin, for example the sequence of albumin human, well known to specialists in this field (see, for example, U.S. patent No. 5876969, which is fully incorporated into the present description by reference). Albumen sequence that can be used for the purposes of this modification of proteins include, for example, pre-Pro-form, full-length form, or fragments thereof (see, e.g., Kratz et al., Arch. Pharm. (Weinheim) 331: 47-53 (1998); Syed et al., Blood 89: 3243-3252 (1997); Makrides et al., J. Pharmacol. Exp. Ther. 277: 534-42 (1996); Breton et al., Eur. J. Biochem. 231: 563-69 (1995); Paige et al., Pharm. Res.12: 1883-88 (1995)). Albumen sequences can be added to the polypeptides according to the present invention during chemical synthesis or by using recombinant methods, or using combinations thereof.

In one non-limiting example, a full-size human albumin can be fused in reading frame with T (AU-TALLEQAQIQQEKNEYELQKLDK-NH2when using recombinant technology.

In the other is a non-limiting example T (AU-TALLEQAQIQQEKNEYELQKLDK-NH 2), which was pagalilauan may be anywhereman with a truncated human albumin (available from Sigma Chemical, St. Louis) using known in the art methods such as the method Paige et al. (Paige et al., Pharm. Res. 12: 1883-88 (1995)).

Modified polypeptides can be tested for biological activity, such as antiviral activity, using standard methods. In addition, using conventional procedures can be analyzed features such modified polypeptides, as pharmacokinetic or immunogenic properties. Modified polypeptides can also be investigated in vitro and in vivo to detect changes in biological reactions associated with the modification(s).

It should be understood that the peptides listed in Table 2 and in the example below in section 11, are also covered by the scope of the present invention. As noted earlier, those of the peptides listed in Table 2 and in the example below in section 11, and which do not contain the enhancer peptide sequence (i.e. which are not hybrid polypeptides), may be used in combination with the enhancer peptide sequences and in the present description of the disclosed methods of obtaining hybrid polypeptides. In addition, core polypeptides and a core polypeptide in the composition of the hybrid polypeptides shown in Table 2, Fig and in the example presented below in section 11, can be used in conjunction with any specified in the description of the enhancer peptide pic what ecovitality the purpose of obtaining, using routine procedures additional hybrid polypeptides, also included in the scope of the present invention.

For example, the peptide DP397 shown in the example of section 11 refers to a core polypeptide and is also included in the scope of the present invention. In addition, a hybrid polypeptide comprising a core polypeptide DP397 in combination with one or more enhancer polypeptide sequences given in this description are also included in the scope of the present invention.

It should be noted that although in Table 2 and Fig are lots of polypeptides with modified amino acids, for example, with blocked amino and/or carboxy-ends or with the inclusion of d-isomers of amino acids (denoted as residues enclosed in brackets), however, any polypeptide comprising a primary amino acid sequence, such as shown in Table 2 and Fig, is also included as part of the scope of the present invention.

Core polypeptide sequence, in particular shown in Table 2, Fig and below in section 11 of example, and hybrid polypeptides comprising such core polypeptides can exhibit antiviral and/or antipathogen activity and/or may have the ability to modulate intracellular processes involving folded spiral the s peptide structure. In addition, such peptides can also be used partly in methods of screening of compounds, including peptides, to detect such activity. Among crustal polypeptide sequences include, for example, those derived from the protein sequences of individual viruses. Among crustal polypeptide sequences also include, for example, amino acid sequence derived from the protein sequences of more than one virus (for example, a core polypeptide derived from HIV-1, HIV-2 and SIV).

In addition, these core polypeptides can represent the above amino acid substitution, deletion and/or insertion for a polypeptide enhancer sequences. In cases where a core polypeptide exhibits antiviral and/or antipathogen activity, such modifications preferably not correct (either by itself or as part of a hybrid polypeptide) specified activity.

In relation to amino acid deletions preferably, in order to produce a core polypeptide contains at least 4-6 amino acid residues in length. In relation to amino acid insertions preferred insertion should not be more than about 50 amino acid residues, and more preferably not more than about 15 amino acid OST tcov. Preferably also, to insert the crustal polypeptide represented amino and/or carboxy-terminal insert.

The number of amino acid substitutions, deletions and/or insertions crustal or hybrid polypeptides according to the present invention include those that correspond to the substitutions, deletions and/or insertions which are detected in mutant forms, for example in natural mutant forms of endogenous protein sequence from which specific core polypeptide was obtained.

For example, if a core polypeptide derived from a viral protein and the specified core polypeptide (either by itself or as part of a hybrid polypeptide) exhibits antiviral activity against one or the other of the virus, it is possible that there may be or may appear eventually such variants of the virus (for example, variants of strain), which have some level of resistance to the peptide in comparison with the level of antiviral effect of the indicated peptide in relation to viral strain, from which was obtained the original endogenous crust polypeptide sequence.

To obtain crustal polypeptides which exhibit antiviral activity against these resistant viral strains may be introduced modifications in the original core polypeptide. In h is particularly any specialist with the average level of knowledge in this field using standard methods can be easily selected isolates of resistant virus. This may be easily carried out sequencing inside a resistant virus, the corresponding source korovou the polypeptide, followed by comparison with the original crustal polypeptide.

In the case when the corresponding sequence derived from a mutant resistant strain is different from the crustal sequence of the polypeptide, and modification of the core polypeptide can be introduced so that the resulting modified core polypeptide had the same sequence as the plot in a stable virus.

The obtained modified core polypeptide either by itself or as part of a hybrid polypeptide will exhibit antiviral properties against viral strain that was resistant to the original korovou the polypeptide. In this regard, these methods can be used to identify crustal polypeptides exhibiting antiviral activity against viral strains that are resistant or purchased specified resistance to the antiviral activity of other crustal polypeptides.

Below in the example section 11 describes one particular, ogranichivayutsya variant of this method for obtaining modified crustal polypeptide, which exhibits antiviral activity against viral strains that are resistant to the "parent" korovou the polypeptide.

In one embodiment of the invention, these modified core polypeptides which exhibit antiviral activity against strains resistant "parent" korovou the polypeptide include, which are introduced amino acid substitutions, deletions and/or insertions, modifying the "parent" of a core polypeptide in such a way that in the resulting modified crust the polypeptide is removed consensus sequence for N-glycosylation and O-glycosylation sites present in the "parent" of the crust polypeptide.

For example, the consensus sequence for N-glycosylation is an N-X-S/T, where S/T stands for either serine or threonine, and X denotes any amino acid except Proline or aspartic acid. Thus, in one embodiment of the invention the parent core polypeptide containing such a consensus sequence can be modified by insertions, substitutions and/or deletions of amino acids, so modified the crust specified polypeptide consensus sequence is deleted.

Among these amino - and carboxy-concave the inserts include, which include the amino acid sequence located at the amino and/or carboxy-ends relative to the endogenous protein sequence, from which was obtained a core polypeptide. For example, if a core polypeptide derived from a protein Dr, this box should include amino and/or carboxy-terminal box containing the amino acid sequence Dr adjacent to the cow polypeptide sequence Dr. The size of such amino and/or carboxy-terminal inserts varies and typically is about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acid residues in the direction of amino - and/or carboxy-end of the original crustal polypeptide.

Hybrid polypepide sequence according to the present invention may also include additional modifications that make it easy to detect the polypeptide. For example, the hybrid polypeptide can be labeled, either directly or indirectly. Methods for labeling peptides are well known to specialists in this field and include, but are not limited to the above list, radioactive, fluorescent, and colorimetric methods. Methods indirect labeling is also well known and include, but are not limited to the above list, marking the Biotin/streptavidin and indirect staining using an is Icel.

The invention also relates to the combination of enhancer polypeptide sequences with the types of molecules other than peptides. For example, the enhancer peptide sequence can be combined with nucleic acid molecules (e.g., DNA or RNA) or with any type of small organic molecules to enhance the pharmacokinetic properties of these molecules.

5.2. Synthesis of peptides

Enhancer, crustal and hybrid polypeptides according to the present invention can be synthesized or obtained using procedures known in the field. See, for example, guidance Creighton (Creighton, 1983, Proteins: Structures and Molecular Principles, W.H.Freeman and Co., NY), which is fully incorporated into the present description by reference. Hybrid polypeptides can be obtained using standard stepwise synthesis in solution or in the solid phase, and fragment condensation or methods Fmoc or Side chemistry (see, e.g., Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., 1997, CRC Press, Boca Raton, Florida and is contained in the link operation; and Solid Phase Peptide Synthesis: A Practical Approach, Atherton &Sheppard, Eds., 1989, IRL Press, Oxford, England and is contained in the link operation). Similarly, it can be carried out the synthesis with modifications at the amino and/or carboxy-ends.

In General, these methods may include the sequential addition of one or more of the am is nakilat or suitably protected amino acids to a growing peptide chain. In norm or amino or carboxyl group of the first amino acid protecting with a suitable protective group. Protected or derivational amino acid can then be either attached to an inert solid substrate, or may be used in the form of a solution by adding the next amino acid in the sequence contains complementary (amino or carboxyl) group, protected in an appropriate manner, the conditions conducive to the formation of amide linkages. Then the protective group is removed from the specified, the newly added amino acid residue, then add the next amino acid (suitably protected) and so on. After all the desired amino acids are linked in the proper sequence, remove the remaining protective group and any solid substrate either sequentially or simultaneously with the process of creating the desired target polypeptide. With the introduction of a simple modification of the published procedure it can be applied to add more than one amino acid, the building chain, for example, when linking (in conditions not conducive racemization of chiral centers) protected Tripeptide protected adequately by the dipeptide with education, after removal of the protective groups, the Penta is eptide.

Typical protective groups include T-butyloxycarbonyl (Side), 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), p-toluensulfonyl (CBT), 2,4-dinitrophenyl, benzyl (Bzl), biphenyltetracarboxylic, cyclohexyl, isopropyl, acetyl, o-nitrophenyloctyl and other of the following groups are preferred Barefoot and Fmoc.

Typical solid carriers are mainly in the cross-crosslinked polymeric materials. They, in turn, include, but are not limited to the above list, cross-stitched divinylbenzene polymers based on styrene, for example copolymers of divinylbenzene-hydroxymethylbutyrate, copolymers of divinylbenzene-chloromethylstyrene and copolymers of divinylbenzene-benzhydrylpiperazine. Such copolymers have the advantage associated with the direct introduction of the terminal amide functional groups in the peptide chain, so that the function of this group is stored in the circuit after the circuit is separated from the base.

This way can be synthesized polypeptides containing either L-or D-amino acids.

The composition of the polypeptide can be confirmed by quantitative amino acid analysis and the presence of specific sequences in each peptide can be determined using the methods used for sequence analysis.

EN Ansary, the core and hybrid polypeptides according to the present invention can be purified by any known in the art methods such as high performance liquid chromatography in normal phase or treatment phase, ion-exchange chromatography, gel electrophoresis, affinity chromatography, particle size distribution method, sedimentation, and other Actual conditions used for the purification of a specific polypeptide, depend, in particular, from the strategy of synthesis and on factors such as charge, hydrophobicity, hydrophilicity, solubility, stability and other parameters, obvious to a person skilled in this field.

Hybrid, enhancer and core polypeptides can also be obtained using recombinant DNA methods. In the present invention the nucleotide sequence encoding the polypeptides according to the present invention, can be synthesized and/or cloned and then expressed in accordance with well-known experts in this field procedures. See, for example, the direction of Sambrook (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Press, NY).

The DNA fragment encoding the desired polypeptide can be obtained using a variety of molecular biology techniques known to experts in this field. For example, for the formation of a DNA fragment encoding be interested in is OK can be used polymerase chain reaction (PCR).

Alternative DNA fragment can be obtained from a commercial source.

DNA encoding the desired polypeptides can be introduced using recombinant technology in a variety of host-vector systems that allow large-scale DNA replication. Can be designed such vectors, which contain the necessary elements for implementation of targeted transcription and/or translation of the DNA sequence that encodes a hybrid polypeptide.

Vectors that may be used include, but are not limited to the above list, vectors derived from recombinant DNA bacteriophage, plasmid DNA or kosmidou DNA. So, for example, can be used plasmid vectors, such as vectors series pcDNA3, pBR322, pUC 19/18, pUC 118, 119 and M13 mp. The bacteriophage vectors may include vectors of bacteriophages series λgt10, λgt11, λgt18-23, λZAP/R and EMBL. Cosignee vectors that can be used include, but are not limited to the above list, pJB8, pCV 103, pCV 107, pCV 108, pTM, pMCS, pNNL, pHSG274, COS202, COS203, pWE15, pWE16 and choroid 9 series vectors.

Alternatively, genetic methods can be created recombinant vectors of viruses that include, but are not limited to the above list, vectors derived from VIR the owls, such as herpes virus, retrovirus, virus, cowpox virus, adenoviruses, adeno-associated virus, or the virus of papilloma of the bull and plant viruses such as tobacco mosaic virus and baculovirus.

For the purposes of expression of a biologically active polypeptide to a nucleotide sequence encoding a protein, can be inserted into the appropriate expression vector, for example, a vector which contains the necessary elements for the transcription and translation of the inserted coding sequences. This can be applied to methods known in the art to construct expression vectors containing a sequence encoding a hybrid polypeptide that is functionally connected with the respective signals, regulation of transcription/translation. These methods include the use of in vitro recombinant DNA and synthetic methods. See, for example, Sambrook et al., 1992, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. and Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates &Wiley Interscience, N.Y., each of which is fully incorporated into the present description by reference.

The nucleic acid molecule encoding a interesting hybrid, enhancer and core polypeptides, may be functionally linked to a variety of different promoter/enhancer elements. These promoter/enhanse the data elements can be selected for optimization of expression of therapeutic amounts of protein. The elements of expression of these vectors can vary by activity and specificity. Depending on the nature of the used host/vector system can be used by any one element of the set of matching items transcription and translation. The promoter may be the promoter, which in its natural state is in communication with the gene of interest. Alternatively, DNA may be under the control of a recombinant or heterologous promoter, i.e. a promoter, which normally is not associated with the specified genome. So, for example, tissue-specific promoter/enhancer elements can be used for regulating the expression of DNA transferred into cells of a specific type.

Examples of areas of regulation of transcription, characterized by tissue specificity, which are described in the literature and may be used include, but are not limited to the above list, the site of regulation of gene elastase I, active in botryoidal the pancreatic cells (Swift et al., 1984, Cell 38: 639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50: 399-409; MacDonald, 1987, Hepatology 7: 42S-51S); the area of regulation of the insulin gene, active in the beta cells of the pancreas (Hanahan, 1985, Nature 315: 115-122); plot the regulation of immunoglobulin gene, active in lymphoid cells (Grosschedl et al., 1984, Cell 38: 647-658; Adams et al., 1985, Nature 318: 533-538; Alexander et al., 1987, Mol. Cell Biol. 7: 136-1444); plot the regulation of albumin gene, active in liver (Pinkert et al., 1987, Genes and Devel. 1: 268-276); plot the regulation of the gene alpha-fetoprotein, active in liver (Krumlauf et al., 1985, Moll. Cell Biol. 5: 1639-1648; Hammer et al., 1987, Science 235: 53-58); plot the regulation of the gene alpha-1-antitrypsin, is active in liver (Kelsey et al., 1987, Genes and Devel. 1: 161-171); plot the regulation of beta-globin gene, active in myeloid cells (Magram et al., 1985, Nature 315: 338-340; Kollias et al., 1986, Cell 46: 89-94); the area of regulation of gene myelin basic protein, active in oligodendrocyte cells of the brain (Readhead et al., 1987, Cell 48: 703-712); plot the regulation of gene light chain 2 myosin active in skeletal muscle (Shani, 1985, Nature 314: 283-286), and plot the regulation of gene gonadotropin, a hormone that is active in the hypothalamus (Mason et al., 1986, Science 234: 1372-1378). Can be used promoters isolated from the genome of viruses grown in mammalian cells (for example, promoters of vaccinia virus C, SV40, HSV, adenovirus MLP, MMTV, LTR and CMV), and promoters derived using recombinant DNA and methods of synthesis.

In some cases, promoter elements can be a constitutive or inducible promoters can be used in appropriate circumstances to achieve expression at a high level or to carry on regulated expression of interest nucleotide sequence. Expression of genes con shall be under the control of constitutive promoters, does not require a specific substrate for the induction of gene expression and occurs when all the conditions of cell growth. Conversely, expression of genes controlled by inducible promoters, responds to the presence or absence of inducer.

For the implementation of the adequate translation of the inserted sequences encoding the protein, also require specific triggering signals. These signals include the initiating ATG codon and adjacent sequences. In those cases, when appropriate expression vectors enter the full coding sequence, including the initiating codon and adjacent sequences, may not need additional signals for regulation of translation. However, in cases when enter only part of the coding sequence, it must be ensured the presence of exogenous broadcast signal, including the initiating codon ATG. In addition, the initiating codon must be in the same phase with the reading frame sequence encoding a protein, to ensure translation of the entire insert. These exogenous signals regulation of translation initiator codons can have a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced with the inclusion of sequences, relaxing the x transcription, enhancer elements and other

5.3. The use of the enhancer peptide sequences, crustal polypeptides and hybrid polypeptides according to the present invention

As mentioned above, the enhancer peptide sequence according to the present invention can be used to enhance the pharmacokinetic properties of any crustal polypeptide by binding crustal polypeptide with the enhancer peptide sequences with the formation of the hybrid polypeptide. The observed enhancement of pharmacokinetic properties is assessed in relation to the pharmacokinetic properties of one crustal polypeptide. A standard set of characteristic pharmacokinetic parameters and methods of determination and evaluation of the pharmacokinetic properties of the agent, such as a polypeptide known to specialists in this field of technology. Non-limiting illustrative variations of such methods are given below in the "Examples"section.

Enhancer peptide sequence according to the present invention may also be used to enhance in vitro or ex vivo half-life of the crustal polypeptide, to which is attached enhancer peptide sequence. For example, the enhancer peptide sequence can increase the half-life of attachable crustal polypep the species if the resulting hybrid, polypeptides in cell culture, tissue culture, or in samples taken from the patient (for example, in samples of cells, biopsy tissue samples or other samples containing body fluids).

Core polypeptides and hybrid polypeptides according to the present invention can also be used as part of methods aimed at modulating (e.g., reduction, inhibition, destruction, stabilization or gain) effects of the merger. Preferably, such peptides are antipathogen or antiviral activity. The peptides according to the present invention may also have the ability to modulate intracellular processes involving interaction folded helical peptides.

In certain embodiments the invention, the hybrid polypeptides and core polypeptides according to the present invention which exhibit antiviral activity, can be used as part of methods aimed at reducing viral infection. Such methods antiviral actions can be applied, e.g., against human retroviruses, in particular HIV (human immunodeficiency virus), such as HIV-1 and HIV-2, and T-limfozitah of human viruses (HTLV-I and HTLV-II), and retroviruses of other organisms other than humans, such as bovine leukosis virus, viruses sarcoma and leiko is and cats viruses human immunodeficiency monkeys (SIV), a virus sarcoma and leukemia viruses progressive pneumonia of sheep.

How antiviral action in accordance with the present invention can also be used against other viruses other than retroviruses, including, but not limited to the above examples, respiratory syncytial virus (RSV), a virus plague dogs, the virus pseudocode birds, parainfluenza virus human influenza viruses, measles viruses, viruses of the Einstein-Barr, hepatitis b viruses and viruses Mason-Pfizer.

The above viruses are viruses containing shell. Antiviral methods according to the present invention can also be applied against viruses that do not contain membrane, including, but not limited to the above list, picornaviruses, such as polio viruses, hepatitis a virus, enterovirus, Echovirus and Coxsackie virus, Papa-babirusa, such as the human papilloma virus, parvoviruses, adenoviruses and reoviruses.

Other types antipathogenic activity that can be modulated using the methods of using the peptides according to the present invention, include, but are not limited to, modulating the metabolism of neurotransmitters through cell fusion and fusion-type sperm-egg. Among the intracellular violations, including interaction folded spiral with whom rector, which can be attenuated using methods of using the peptides according to the present invention, include, for example, diseases associated with the action of bacterial toxins.

Antimutagenna or antiviral activity of this crustal polypeptide or hybrid polypeptide can be readily established by standard methods of in vitro, ex vivo using animal models, and in regard to the determination of antiviral activity, they can be specific or part of specific interest to the virus, and they are all well known to specialists in this field.

The above description refers primarily to antiviral activity and activity-related antimutagennym effect of the crustal and hybrid polypeptides according to the present invention. Hybrid polypeptides according to the present invention can also be used as part of any method, which can be considered the introduction of, or other use of one crustal polypeptide. The use of hybrid polypeptides as an integral part of these methods is particularly preferable in cases where it is desirable to increase the pharmacokinetic properties of the crustal polypeptide. So, for example, insulin is used as a component of treatment when n is which types of diabetes. In this regard, as an integral element methods aimed at relieving symptoms of those forms of diabetes, in which the use and/or want to use insulin, you may use a hybrid polypeptide comprising an insulin or a fragment of insulin as the crustal polypeptide.

In addition to the above methods, the peptides according to the present invention can also be used as an integral part of predictive methods aimed at preventing diseases, including, but not limited to, diseases associated with the merger process, intracellular processes involving collapsed helical peptides, viral infection, in which there is a merger-type cell-cell and/or virus-cell. For example, the core and hybrid polypeptides according to the present invention can be used in the prevention of methods used to prevent viral infections.

Hybrid polypeptides according to the present invention can also be used as part of diagnostic methods. Such methods can refer either to in vitro methods, or methods in vivo. Any diagnostic method, which can be used specific core polypeptide, can also be applied using a hybrid polypeptide, vkluchaya what about the core polypeptide and modification of the primary amino acid sequence, detecting the hybrid polypeptide. Such techniques reflect the improvement of diagnostic methods that are defined by the fact that the increased half-life of the hybrid polypeptide in comparison with the crustal polypeptide can lead to increased sensitivity of the diagnostic procedure in which it is used. These diagnostic procedures include, but are not limited to, imaging techniques, for example, imaging techniques in vivo. In a non-limiting example, imaging techniques, the structure that connects the core polypeptide hybrid polypeptide can be detected by binding hybrid polypeptide and visualization (either direct or indirect) associated to the hybrid polypeptide.

5.4. Pharmaceutical compositions, dosage and routes of administration

The peptides according to the present invention can be introduced using known in the art methods. Preferably the considered tools include in the composition and is administered systemically. The procedures used for preparation of compositions and their administration, are described, in particular, the manual Remington ("Remington''s Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton, PA). Suitable routes of administration include oral, rectal, endovaginal, intra-lungs (for example, by ingal the tion), cutaneous, crosslisted or intracolonic introduction, parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as vnutriobolochechnoe directly intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, to name a few options. In the case of intravenous injection means according to the present invention can be manufactured in the form of aqueous solutions, preferably in physiologically compatible buffers such as Hanks solution, ringer's solution, or physiological saline, to name a few options. In addition, for the delivery of peptides according to the present invention can be applied infusion pumps. For crosslisted introduction in the manufacture of a composition used amplifiers permeability, suitable for barrier through which it should pass. Such amplifiers permeability generally known in the art.

In cases where preferably the intracellular introduction of the peptides according to the present invention, or other inhibitors that can be applied to methods known in the art. For example, these funds can be encapsulated in liposomes or microspheres and then entered using the above methods. Liposomes are spherical l is penye bikinie particles with internal water environment. All the molecules present in the aqueous solution for formation of liposomes, are included in the internal aqueous environment. Liposomes are protected from the external environment, and, due to the fact that liposomes fused with the cell membrane, are able to effectively reach the cell cytoplasm. In addition, due to the hydrophobicity of liposomes in the case of small molecules can be achieved direct intracellular injection.

The nucleotide sequence encoding the peptides according to the present invention when their intracellular delivery, can be expressed in cells of interest using known in the art methods. For example, expression vectors derived from viruses such as retroviruses, viruses cow pox, adeno-associated viruses, herpes viruses, or viruses of a papilloma of the bull, can be used for delivery and expression of such nucleotide sequences in a population of target cells. Methods of constructing such vectors and expression constructs also known. See, for example, Sambrook, et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY and Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates &Wiley Interscience, N.Y.

Effective doses of the peptides according to the present invention, which should be entered, can be determined using known in the art procedure is, taking into account such parameters as biological half-life, bioavailability and toxicity. In especially preferred embodiments of the invention, the range of effective dosages of the hybrid polypeptide can be determined by any person skilled in the art using routine in vitro and in vivo, are known in the art. For example, the evaluation of the antiviral activity in cell culture in vitro using methods, examples of which are given below in section 7 for T, provides data on the basis of which any person skilled in the art can easily determine the average inhibitory concentration (IR) peptide according to the present invention, necessary to block a certain level of viral infectivity (e.g., 50% IR50or 90% IR90). And then the appropriate dosage can be chosen by any person skilled in the art based on the pharmacokinetic data obtained using one or more conventional animal models, such as pharmacokinetic data described below in the examples section 10 for T, obtaining the value of the minimum plasma concentration (Smin) peptide, which is equal to or exceeds a certain value IR.

Approximate doses of the polypeptide can include a low dose such as 0.1 μg/kg body weight, and high doses, such as 10 mg/kg of body weight. More preferably, the range of effective doses ranging from 0.1 to 100 μg/kg of body weight. Other illustrative examples of dosages of the peptides according to the present invention include 1-5 mg 1-10 mg, 1-30 mg, 1-50 mg, 1-75 mg, 1-100 mg, 1-125 mg, 1-150 mg, 1-200 1-250 mg or mg of peptide. A therapeutically effective dose refers to an amount of compound which is sufficient to alleviate symptoms or to increase survival of the patient. Toxicity and therapeutic efficacy of such compounds can be determined using standard pharmaceutical procedures in cell cultures, experimental animals, for example, when defining LD50(lethal dose for 50% of the population) and ED50(the dose therapeutically effective in 50% of the population). The ratio of doses that cause toxic and therapeutic effects is therapeutic index, which can be expressed as the ratio LD50/ED50. Preferred compounds with high therapeutic index. The data obtained through such analysis methods cell cultures and animal studies, can then be used to establish dosages when used on people. The dosage of such compounds lies preferably within a range of circulating blood concentrations, which which include ED 50with little toxicity or no toxicity. The dosage may vary within this range depending on the form used, the dosage and the way of its introduction. A therapeutically effective dose of any of the compounds used in the framework of the method according to the present invention, first assess the results of the analysis of cell culture. Further, animal models can be defined dose required to achieve a range of circulating plasma concentrations, which includes IR50(for example, the concentration of tested compound that achieves a half of the maximum level of inhibition fusogenic effect, such as half of the maximum level of inhibition of viral infection in comparison with the level of this phenomenon in the absence of the tested compound)determined in cell culture. Such information can be used to provide a more accurate determination of dosages for use in humans. The concentration levels in plasma can be determined, for example, by high performance liquid chromatography (HPLC) or any biological or immunological method to measure the levels of peptides.

Hybrid polypeptides according to the present invention can be administered as a single dose, fractional, and periodic and continuous reception. For example, the polypeptides according to the present invention can be administered as a single dose, such as a single subcutaneous, single intravenous infusion or a single ingestion. The polypeptides according to the present invention can be represented using many forms of fractional introduction, including periodic introduction. For example, in some embodiments, the polypeptides according to the present invention can be administered once a week, once a day, twice a day (every 12 hours), every six hours, every four hours, every two hours or every hour. The polypeptides according to the present invention can be dened for continuous administration, such as continuous subcutaneous or intravenous infusion using a pump or by using a subcutaneous or other implant, which allows continuous suction of the polypeptides in the patient's body.

Hybrid polypeptides according to the present invention can also be administered in combination with at least another therapeutic agent. Although this option is not preferred in the treatment of HIV, in the case of the treatment of other diseases (e.g. cancer treatment) introduction can be carried out in concomitant or sequential mode, including cyclic regimen (which includes the introduction is the first connection during a certain period of time, then the introduction of a second antiviral compounds within a certain period of time and repeating the sequential introduction in order to reduce the development of tolerance arising to a single type of treatment).

In case of a virus, for example, retroviral, infection can be introduced effective amount of the hybrid polypeptide or its pharmaceutically acceptable derivative in combination with at least one, preferably at least two, other antiviral agents.

If we consider the example of HIV infection, the antiviral agents may include, but are not limited to the above examples, DP-107 (T21), DP-178 (T20), any other core polypeptide shown in Table 2, which was obtained from HIV-1 or HIV-2, or any other hybrid polypeptide, a core polypeptide which is at least partially derived from HIV-1 or HIV-2, cytokines, for example, rIFN α, rIFN β, rIFN λ, reverse transcriptase inhibitors, including nucleoside and non-nucleoside inhibitors, such as AZT, FTC, D4T, ddI, adefovir, abacavir and other dideoxynucleoside or dimethoxyphenylacetic or mesilate of delavirdine, nevirapine, efavirenz, inhibitors capping viral mRNAs, such as ribavirin, inhibitors of HIV protease, such as ritonavir, nelfinavir mesilate, APV,SQV, saquinavir mesilate, indinavir or AWT, AWT or MC, amphotericin b as communicating with the lipid molecule with anti-HIV activity, and castanospermine as an inhibitor of glycoprotein processing.

Hybrid and/or core polypeptides according to the present invention can also be used prophylactically to prevent the development of disease. Hybrid and/or core polypeptides can prevent the disease due to their direct action or alternative can be used as vaccine, when in the host body increases production of antibodies against hybrid polypeptides according to the present invention, which then serve to neutralize pathogenic organisms, including, for example, inhibition of viral, bacterial and parasitic infections.

For all of these treatments specific type of composition, the way of its introduction and dose are selected by the attending physician based on the patient's condition (see, e.g., Fingl et al., 1975, in "The Pharmacological Basis of Therapeutics, Ch.1, p.1).

It should be noted that the attending physician must know how and when to end, suspend or amend the introduction, in the case of toxicity or organ dysfunction. Conversely, the attending physician should also be aware of the need to adjust treatment by increasing the input voltage is ima dose if there is inadequate clinical effect (without causing toxicity). The magnitude of the injected dose in the treatment of oncogenic disease varies depending on the severity of the condition to be treated and the route of administration. Dose and possibly frequency of the dose will also depend on the age, body weight and individual response of the patient. The program, comparable to that discussed above, can also be used in veterinary medicine.

The use of pharmaceutically acceptable carriers to create compositions based on the compounds disclosed in the present description, in the form of particular dosage form suitable for systemic administration, is also included in the scope of the present invention. When choosing the appropriate media and using appropriate technology for the production of the composition according to the present invention, in particular, those that are made in the form of solutions, may be administered parenterally, by using techniques such as subcutaneous injection, intravenous injection, subcutaneous infusion or intravenous infusion, for example, using an infusion pump. Using known pharmaceutically acceptable carriers compounds can be easily prepared in the form of a composition in a dosage that is acceptable for oral administration. These media allow the manufacture of the as the basis of the compounds according to the present invention such forms, as tablets, pills, capsules, liquid formulations, gels, syrups, shaped in the form of a slurry, suspension, etc. for oral administration by the patient undergoing treatment.

Pharmaceutical compositions suitable for application in the framework of the present invention include compositions where the active ingredients are contained in an effective amount sufficient to achieve the objectives. Determination of the effective amounts is able to hold any specialist in this field, especially in connection with the detailed description of the present invention.

In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and additives that facilitate the manufacturing process on the basis of the active compounds of preparations which can be used for pharmaceutical purpose. Drugs, prepared for oral administration may be in the form of tablets, pills, capsules or solutions. For oral administration of peptides can be used in the procedure, such as, for example, is practiced in Emisphere Technologies, and which is well known to specialists in this field.

The pharmaceutical compositions according to the present invention can be produced using methods which are in themselves well known, for example, when implementing the traditional processes of mixing, dissolving, granulating, get pills, grinding into powder, spray drying, emulsification, encapsulation, capture or freeze-drying.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. In addition, emulsions and suspensions of the active compounds can be prepared as appropriate oily mixtures for injection. Suitable lipophilic solvents or carriers include fatty oils such as sesame oil, or synthetic fatty acid esters, such as etiloleat, or triglycerides, liposomes, or other known in the art substances used to obtain a lipid or lipophilic emulsions. Aqueous suspension for injection may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Suspension may also optionally contain suitable stabilizers or tools that enhance the solubility of the compounds, allowing for highly concentrated solutions.

Pharmaceutical compositions for oral administration can be obtained by combining the active compounds with solid excipient, optionally grinding the resulting mixture and processing the mixture of granules, after adding, if desired sootvetstvuyushchego, to obtain tablets or dragee cores. Acceptable carriers are, for example, fillers such as sugars, including lactose, sucrose, trehalose, mannitol or sorbitol, cellulose preparations such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragakant, methylcellulose, hydroxypropyl-methylcellulose, sodium carboxymethylcellulose and/or polyvinyl pyrrolidone (PVP). If desired, can be added to contribute to decomposition, such as cross-crosslinked polyvinylpyrrolidone, agar or alginic acid or its salt, such as sodium alginate.

At the core of drops put the appropriate coverage. For this purpose, can be used in concentrated sugar solutions, which may not necessarily contain the Arabian gum, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and/or titanium dioxide, lakiruyuschii solutions and suitable organic solvents or mixtures thereof. The tablets or covering shells drops can be added dyes or pigments for identification or to indicate different combinations of doses of the active ingredient.

Pharmaceutical preparations which can be used orally include capsules notch, made of gelatin, and also soft sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Capsules notch may contain active ingredients mixed with a filler, such as lactose, binders, such as starches, and optionally a stabiliser. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Additionally may be stabilizers.

In cases where it is desirable to increase the immune response of the host, the hybrid polypeptides can be prepared in the form of a composition in combination with a suitable adjuvant for the purposes specified strengthen the immune response. Such adjuvants may include, but are not limited to the above list, mineral gels such as aluminum hydroxide; surface active substances such as lysolecithin, planovye of polyole, polyanion; other peptides; oil emulsions, and potentially useful adjuvants such as BCG and Corynebacterium parvum. For the introduction of the vaccine compositions used many methods. These methods include, but are not limited to, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous and intranasal routes of administration.

6. Example: Identification of consensus amino acid sequences that include the enhancer Pat the derivative sequence

Protein of the retrovirus Dr contains structural domains related to the site α-helix located in the C-terminal region of the protein, and a section of "zipper", located in the N-terminal region of the protein. Comparison plots enhancer peptide sequence contained within Dr (figa and 2B), in Dr of all selected sequences of HIV-1, HIV-2 and SIV, the publication of which has so far allowed us to identify a consensus amino acid sequence shown in figure 1.

As will be described in more detail in the examples below, these sequences represent the enhancer peptide sequence in the specified data binding peptide sequences with many different crustal polypeptide that enhances the pharmacokinetic properties of the obtained hybrid polypeptides.

7. Example: Hybrid polypeptides that function as potent inhibitors of HIV-1 infection

As shown in Fig, T is a hybrid polypeptide comprising enhancer peptide sequence associated with crustal HIV polypeptide. As will be shown below, the hybrid polypeptide T shows enhanced pharmacokinetic properties and potent activity in vitro against isolates of HIV-1, HIV-2 and SIV, and show improved the activity against clinical isolates of HIV-1 tests for infectivity in relation huPBMC in vitro, and huPBMC with SCID (severe combined immunodeficiency) mouse model of HIV-1 infection in vivo. In the following biological tests activity T compare with powerful antiviral polypeptide T20. The T20 polypeptide, also known as DP-178, obtained from the protein sequence Dr HIV-1, which was described and claimed in U.S. patent No. 5464933.

7.1. Materials and methods

7.1.1. Synthesis and purification of peptides

Peptides are synthesized using the methods Fast Mos chemistry. Basically, unless explicitly noted, the peptides contain amidarone carboxyl ends and acetylated amino ends. Cleaning is performed by HPLC with treatment phases.

T (Ac-WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF-NH2) is a peptide of 39 amino acids (MV=5036,7), consisting entirely of naturally occurring amino acids and blocked at the amino end of the acetyl group, and the carboxyl end of the amido group with the aim of increasing stability. T is a peptide consisting of 23 amino acids and containing no enhancer peptide sequence AC-TALLEQAQIQQEKNEYELQKLDK-NH2). Thus, T is a core polypeptide hybrid polypeptide T. T blocked by its amino - and carboxy-ends in the same way as T.

In particular, T synthesized using standard methods of synthesis in the solid phase. Identichnost the main peak in the HPLC profile T confirmed by the data of mass spectroscopy.

T easily purified by chromatography treatment phase of 6-inch column filled with C18, 10 micron, with the substrate 120A.

7.1.2. Virus

HIV virus HIV-1LAI(Popovic, M. et al., 1984, Science 224: 497-508) propagated in CEM cells cultured in medium RPMI 1640 containing 10% fetal calf serum. Supernatant from infected CEM cells passed through a 0.2 μm filter and estimate the titre of infectivity in microinjection test using cell lines AA to support virus replication. For this purpose add 20 ál of serially diluted virus to 20 μl of CEM cells at a concentration of 6×105/ml on 96-well microtiter tablet. Each dilution of virus examined in three repetitions. Cells cultured for seven days adding every other day with fresh medium. On day 7 after infection, the supernatant samples are tested for the presence of virus replication on the basis of determining the activity of reverse transcriptase inhibitor released from the supernatant. The value TCID50calculated by the formula of reed and Munga (Reed and Muench; Reed, L.J. et al., 1938, Am. J. Hyg. 27: 493-497).

7.1.3 Test merge cells

About 7×104cells Molt-4 incubated with 1×104the CEM cells in chronic HIV virus HIV-1LAI96-hole tablet for tissue culture in culture medium in a total volume of 100 µl (RPMI 640, containing 10% inactivated by heating the FBI with the addition of 1% L-glutamine and 1% penicillin-streptomycin), as described previously (Matthews, T.J. et al., 1987, Proc. Natl. Acad. Sci. USA 84: 5424-5428). Add the inhibitor peptide in a volume of 10 μl and the cell mixture is incubated for 24 hours at 37°C in an atmosphere of 5% CO2. After a specified time count the number mnogokadrovyh giant cells (syncytium, width corresponding to the five cells or more) when using the microscope with magnification of 10× and 40×, which you can explore in the field of view of the entire hole. Treated cells compared to infected untreated controls and the results expressed as percent inhibition of infected control.

7.1.4. Tests for infectivity Magi-CCR-5

About 1×104cells Magi-CCR-5 (obtained from the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID; Chackerian, B. et al., 1997, J. Virol. 71: 3932-3939) were seeded on 48-well plate for tissue culture (approximately 2×104cells/well in selective growth medium in a volume of 300 µl/well, consisting of DMEM with addition of 10% inactivated-tion by heating the FBI, 1% L-glutamine, 1% penicillin-streptomycin, hygromycin, geneticin and puromycin) and leave for contacting overnight at 37°C in an atmosphere of 5% CO2. Merge cells reached approximately 30% following the th day. The sowing medium remove and add the diluted peptide inhibitor in an amount of 50 μl/well (for environments that are made only in untreated controls), and then 100 μl/well of diluted virus (desired input titer of the virus is 100-200 CFU/well). Finally, for each cell add 250 ál of selective growth medium and tablets incubated for 2 days at 37°C in an atmosphere of 5% CO2. Fixation and staining is carried out in accordance with the Protocol of the National Institute of Allergy and infectious diseases (USA), designed for cell MAGI-CCR5. Briefly, the procedure consists in the fact that from the tablet removes the environment and to each well, add 500 ál of fixative. Tablets leave on for 5 minutes at room temperature for fixing. The latch is removed, each well was washed twice in DPBS and to each well add 200 ál of dye solution. Next, the tablets incubated for 50 minutes at 37°C in an atmosphere of 5% CO2the dye solution is removed and each well was washed two times with DPBS. Tablets leave to air dry, after which, under the microscope, examining the whole hole count the number of stained blue cells. The data obtained for the treated cells, compared with those of the infected, untreated controls and the results expressed as percent inhibition infected to the trolls.

7.1.5. Test reverse transcriptase

For determining reverse transcriptase (FROM) in terms of Microtest adapt the procedure Hoffa et al. (Goff, et al., 1981, J.Virol. 38: 239-248) and Willi et al. (Willey, R. et al., 1988, J.Virol. 62: 139-147). In supernatant of virus/cell cultures lead content of Triton X-100 to 1%. To 50 µl of the cocktail (75 mm KCl, 2 mm reagent Cleveland, 5 mm MgCl2, 5 μg/ml poly A, and 0.25 units/ml oligo dt, of 0.05% NP40, 50 mm Tris-HCl, pH of 7.8, 0.5 µm of non-radioactive dTTP and 10 cCi/ml32P-dTTP) on 96-well microtiter tablet with a U-shaped bottom, add 10 ál of each sample supernatant/Triton X-100 and incubated at 37°C for 90 minutes. After incubation, 40 μl of reaction mixture from each well is transferred to the office of Schleicher and Swalla (Schleicher and Schuell, S+S) for dot-blot analysis under partial vacuum containing mesh 96-alopecia separator (Wallac, catalogue number 1450-423) and the filter substrate, saturated 2×SSC buffer (0.3 M NaCl and 0.003 M sodium citrate). Each well was washed 4 times at least 200 ál of buffer 2xSSC at full vacuum. Device for microdelivery disassemble, remove the mesh filter paper and washed 3 times with buffer 2×SSC. At the end of the procedure impregnate the filter membrane absorbent paper, allow it to air dry and sealed in packages, capacative is presented at an elevated temperature. The sample is placed in luminescense the cartridge, making it cleared fluorescent screen for at least 8 min and close. The exhibition lasts for 16 hours. The value of the pixel index (PIV)received in the specified format, the evaluation of fosforo-luminescent blots (Molecular Dynamics Phosphorimager), is then used to determine enabled or inhibited fraction (Fa) for all doses of the inhibitor(s) when compared to untreated, infected controls (analysis performed on the program ImageQuant excluding background values).

7.1.6. Test the infectivity/neutralization by RVMS

In the prototype specified test using cell lines that use RVMS obtained from the International blood Bank (Interstate Blood Bank), activated in 2-3 days using a combination ACTS (0.5 μg/ml) and CD28 antibodies (0.1 mg/ml). Target cells combine with the medium for isolation of lymphocytes (LSM), washed and frozen. When in need of thawed cells and activate, as noted above, for at least 2-3 days prior to testing. In the specified case definition cells take in a concentration of 2×106/ml in 5% IL-2 environment and in the final volume of 100 μl. Prepare a standard peptide solution in DPBS (1 mg/ml). Breeding peptides do in complete medium containing 20% FBI RPMI 1640/5% IL-2.

7.1.7. Model of infection the Oia in vivo HIV-1 using huPBMC and mice SCID

Females of SCID mice (aged 5-7 weeks) injected intraperitoneally 5-10×107RVMS adult. Two weeks after recovery infect mice intraperitoneally on day 0 103TCID50HIV-1 9320 (AZT-sensitive isolate A). Treatment with peptides spend intraperitoneal injection 2 times a day, starting with day 1 and continue up to 6 days. The spread of the infection in the blood cells, splenocytes, lymph nodes and peritoneal cells assessed by co-cultivation with blast monocarbide cells in human peripheral blood weekly for three consecutive weeks followed by bleeding the animals and tissues (day 7, approximately 12-18 hours after the last injection). Supernatant from culture by co-cultivation evaluate from the point of view of education P24 antigen of HIV-1 as an indicator of the development of viral infection (sets Immunotek Coulter and the appropriate Protocol).

7.1.8. Pharmacokinetic studies in rats

Use of male rats CD-line weighing 250-300 g dual catheter in the jugular vein, obtained from Charles river Laboratories (Charles River Laboratories). Peptides injected in the form of peptide solution in a jugular catheter in the amount of 200 μl (about 3.75 mg/ml), the dose of the solution of appropriate concentration determine Aut method Adegoke (Edelhoch, 1967, Biochemistry 6: 1948-1954) and adjust it in accordance with the weight of the animals, so that each animal received a dose of 2.5 mg/kg Taken approximately 250-300 µl of blood in the specified time periods (0, 15, 30 min and 1, 2, 4, 6 and 8 hours) and added to the test tubes with EDTA. The plasma is selected from precipitated by centrifugation of the cells and either freeze or immediately begin to prepare for conducting fluorescence HPLC analysis.

7.1.9. Fluorescent HPLC analysis of plasma samples

100 μl of the plasma sample is added to 900 μl of precipitating buffer (acetonitrile, and 1.0% TFU, detergent) to give the precipitate, which is a large part of the plasma proteins. After centrifugation at 10,000 rpm for 10 minutes take 400 μl of the supernatant and added to 600 μl of water for HPLC. Based on the concentration of peptide in each sample, make a serial dilution dilution buffer, comprising 40% of precipitating buffer and 60% water for HPLC. In addition to the dilution of the sample, make a serial dilution of a solution of the dose in the buffer and in plasma and use them to plot a standard curve connecting the values of the peak area of known concentration of the peptide. The specified curve is then used to calculate the concentration of the peptide in plasma, taking into account all dilutions made and the number entered in the column.

7.1.10. HTT Protocol

To measure ototoksicescoe/cytostatic effects of peptides spend XTT test (Weislow O.S. et al.,1989, J. Natl. Cancer Inst. 81: 577-586) in the presence of varying concentrations of the peptide for the effective determination of the index of selectivity (IP). TK50determine at the specified method when incubating cells in the presence and in the absence of serial dilutions of the peptide followed by the addition of XTT. In survivors/metabolizing cells HTT restored to soluble brown pigment - HTT-formazan. Determine the amount of absorption and compare the values obtained in the presence and in the absence of the peptide to determine the magnitude TK50method Curbera (see, for example, E.H. Lennette et al., Eds., 1969, "Diagnostic Procedures for Viral and Rickettsial Infections", American Public Health Association, Inc., fourth ed., pp.47-52). Cells Molt 4, SEM (80,000 cells/well) and combinations of two types of cells (70000 and 10000, respectively) contribute to microtiter tablet and incubated with serial dilutions of the peptide within 24 hours in the total volume of a mixture of 100 μl. After incubation each well add 25 ál of working standard solution (1 mg/ml XTT, 250 μm FMS in complete medium containing 5% DMSO) and tablets incubated at 37°C. Measure developed staining and the results are used to Express the values obtained in the wells containing peptides, as percentage of the values obtained for wells with untreated controls.

7.2. Results

7.2.1. Antiviral Akti is ness - tests on the merger

Conduct a direct comparison T with T20 in the test for merger-type cell-cell-mediated virus, using chronically infected CEM cells, mixed with uninfected cells Molt-4, as shown below in Table 3. A comparison of the level of inhibition merge T in the presence of laboratory isolates, such as IIIb, MN and RF, with similar results for T20 indicates approximately 2.5-5-fold superiority relative to the first T20. T was also more active (in excess 3-28 times)than T20, against several clinical isolates inducing syncytia, including AZT-resistant isolate (G691-2), the isolate obtained before treatment AZT (G762-3) and 9320 (the isolate used in the research huPBMC-SCID). Most notably, T was 800 times more than the T20 against HIV-2 NIHZ.

1
Table 3
Isolate virusT20 (ng/ml)nT (ng/ml)nThe difference (again)
HIV-1 IIIb2,591,092,5
HIV-1 G691-2 (AZT-R)406,0116,0125
HIV-1 G762-3 (up to AZT)340,1112,228
HIV-1 MN20,073,176
HIV-1 RF6,172,173
HIV-1 9320118,4134,513
HIV-2 NIHZ3610>104,32840

7.2.2. Antiviral activity tests for infectivity for Magi-CCR-5

Tests for infectivity for Magi-CCR-5 allow for a direct comparison of viral isolates inducing and pendulous syncytia, as well as a comparison of laboratory and clinical isolates. The specified test is also a direct indicator of viral infection (expression of TAT after infection, transactivation of education beta-galactosidase under the action of repeated sequences LTR), in contrast to the commonly used indirect measurements of infectivity, such as the production of antigen P24 or reverse transcriptase. Tests for infectivity using Magi-CCR-5 (see Table 4, below) showed that T constantly more effective than T20, against all tested isolates, both from the point of view of the values of EC50and inhibition values Vn/Vo=0,1. T d will show a significant increase in activity against a clinical isolate of HIV-1 301714 (> 25 times), which is one of the most sensitive isolates in relation to T20. T at least 100 times more active than T20 against isolate SIV W. These results together with data merge cells suggest that T is a potent peptide inhibitor of HIV-1, HIV-2 and SIV.

7.2.3. Antiviral activity tests for infectivity for huPBMC

Conduct a direct comparison T with T20 in tests for infectionscinnosti for huPBMC (table 5 below), recognized by this system in vitro studies, which allows to predict the concentration of drug in plasma is necessary to suppress the virus in vivo. These comparisons show that T is a more powerful tool against all isolates of HIV-1 tested to date, all of the values Vn/Vo=0,1 (dose required to reduce the titre of virus in a single logarithm) are reduced to concentrations measured in the frequency range below microgramos values. Many isolates from a number less sensitive to T20 demonstrate a 10-fold or greater sensitivity to T. It should be noted that HIV-1 9320 - isolate used in models of infection mice huPBMC-SCID, 46 times less sensitive to T20 than T, indicating on ICIE good correlation with the results obtained in the test in vivo.

7.2.4. Antiviral activity of lab isolates resistant to T20

Conduct a direct comparison T with T20 in tests on a merge-type cell-cell-mediated virus, using chronically infected CEM cells, mixed with uninfected cells Molt-4 (table 6 below). T was almost 200 times more active than T20, against T20-resistant isolate.

Table 6
Isolate virusT20 (ng/ml)nT (ng/ml)nThe difference (again)
HIV-1 pNL4-3 SM (T20-resistant)405,332,13193

In tests with cells Magi-CCR-5 (see Table 7 below) T was 50,000 times more active than T20, T20 against resistant isolates, such as pNL4-3 SM and pNL4-3 STM (Rimsky L. and Matthews, T., 1998, J. Virol. 72: 986-993).

Conduct a direct comparison T with T20 in tests for infection huPBMC (see Table 8 below) and assess differences in activity against drug-resistant isolates. T more than 250 times, surpasses T20 activity against resistant isolates pNL4-3 SM.

7.2.5. Antiviral activity model in vivo SCID-huPBMC

Antiviral activity T in vivo compared directly with the activity of T20 in the model of HIV-1 9320 using mice with huPBMC-SCID (figure 3). Two weeks after recovery huPBMC mice infect 103TCID50HIV-1 9320 after passages in RWMS (AZT-sensitive isolate A). Treatment with peptides spend intraperitoneal injection twice daily total daily dose of 67 mg/kg (T20), 20 mg/kg (T), 6,7 mg/kg (T), 2.0 mg/kg (T) and 0.67 mg/kg (T) for 8 days, starting 1 day. The spread of the infection in the blood cells, splenocytes, lymph nodes and peritoneal cells assessed by co-cultivation with blast monocarbide cells in human peripheral blood weekly for three consecutive weeks followed by bleeding the animals and tissues (day 7, approximately 12-18 hours after the last injection drugs). Supernatant from culture by co-cultivation evaluate from the point of view of production P24 antigen of HIV-1 as an indicator of the development of viral infection. Infectious virus was not detected in the blood or lymphatic tissues in animals, which were injected T20, although the virus was detected in peritoneal washouts and drug spleen. All segments were negative for the presence of infectious virus at a dose of 6.7 mg/kg T, indicating at least a 10-fold increased activity compared with the results of treatment T20. At a dose of 2.0 mg/kg T and lymph, and spleen were completely free of detectable infectious virus with a rate of decrease in the titre of virus in peritoneal washings 2 log10and the rate of decrease in the titer of virus in the blood 1 log10compared with infected controls. At the lowest dose T is 0.67 mg/kg the results of the analysis of peritoneal swabs and blood were equivalent to those of the infected control, but at least 1 log10the lower titers of infectious virus was observed in the lymph and spleen tissues. In General, the results show that T from 30 to 100 times more active against HIV-1 9320 in vivo under specified conditions.

7.2.6. Pharmacokinetic studies in rats

To further study the pharmacokinetic profile T use rats with cannulas. Male CD rats weighing 250-300 g enter T and T20 intravenously through a catheter in the jugular vein (figa-5). The resulting plasma samples evaluated by fluorescence HPLC to determine the amount of peptide in extracted plasma. The beta phase half-life and the total value of AUC T were almost three times higher than the T20 (figure 5).

7.2.7. Cytotoxicity

As can be seen from Fig.6, not found obvious signs of cytotoxicity T in vito.

In addition, T has no acute toxicity (mortality within 24 hours) at a dose of 167 mg/kg (highest tested dose) when administered intravenously through a cannula in the jugular vein (0.3 ml in 2-3 minutes).

7.2.8. Direct linking design M41Δ178 from Dr

T subjected to radioactive tagging using125I and then hold purification by HPLC until the maximum specific activity. In the same way idiot T20. Spend saturating binding M41Δ178 (truncated protein merge from ectodomain gp41 without amino acid sequences T20)immobilized on microtiter tablets in the amount of 0.5 mg/ml, as shown in Fig.7. Nonspecific binding is defined as binding radioligand in the presence of 1 ám its peptide. Specific binding is the difference between total and nonspecific binding. The results show that125I-T1249 and125I-T20 have similar affiniscape binding at 1-2 nm. Analysis of inverse functions according to the schedule of Scatchard (Scatchard plots) allows to assume that each ligand binds to a homogeneous class of sites.

The kinetics of binding125I T and125I-T20 determined by scintillation tablets for microtitration coated with 0.5 μg/ml 41D178. The period during which the about are the Association and dissociation, shown in Fig. The dissociation of the bound radioligand determined after adding its peptide at a final concentration of 10 μm in one-tenth of the total volume used in the test. Source and achieved as time passes, the speed for125I-T1249 significantly lower than for the125I-T20. The parameters for the dissociation of both ligands do not change, if the dissociation initiate its other peptide (i.e.125I-T1249 with T20).

To further demonstrate the fact that the two ligands compete for the same target site, titrated its T and T20 in the presence of a single concentration of125I-T1249 or125I-T20. The ligand is added immediately after the start of incubation, its peptide. Curves competitive inspirowane shown in figure 9, suggest that although both have similar ligand affinity, requires a higher concentration of its T or T20 for the full replacement125I-T1249 competitive binding.

7.2.9. Direct binding HR1 plot Dr

Use the spectroscopy circular dichroism (CD) to determine the secondary structure of one T in solution (phosphate buffer solution, pH 7) and in combination with a peptide containing 45 residues (T), HR1 (heptanal repeat 1) binding site Dr. On figa shows the range of KD for one T dissolve in the e (10 μm, 1°). Range typical for peptides with alpha-helical structure. In particular, scan the specified range using a single metric decomposition, is analyzed for a set of spectra of 33 proteins, indicates the presumed contents of the helix in the structure T (one solution) up to 50%. On FIGU shows the spectrum T mixed with T. Black squares (▪) denote theoretical option of the spectrum predicted for the case of "models of non-interaction", in which it is assumed that the peptides in solution do not interact. The real spectrum obtained in the experiment (•), significantly different from theoretical version of the spectrum in the model of non-interaction", indicating that two peptide truly interact, the result detected by the measurement of structural changes that are observed in the spectrum of the CD.

7.2.10. Protection from protease binding site T in Dr

The sensitivity of the chimeric protein M41Δ178, described earlier in section 7.2.8, to cleavage by proteinase K, define and analyze by electrophoresis in polyacrylamide gel. The results are presented on Fig.

In the case of a separate incubation or M41Δ178 (raw; Fig, lane 2), or T (raw; Fig, lane 4) with proteinase K (Fig, lanes 3 and 5, respectively the military) are both split. However, in the case when T incubated with M41Δ178 before adding proteinase K (Fig, track 7) revealed reserved HR-1 fragment size of about 6500 daltons. The sequencing of the protected fragment shows that it corresponds to the site of the primary sequence located within ectodomain Dr. The protected fragment contains soluble HR1 peptide (T)used above in section 7.2.9 when studying the spectrum of KD and contains seven amino acid residues located at the amino end. This protection can be determined by linking T with a specific sequence t, which is contained in the design M41Δ178.

8. Example: hybrid polypeptides respiratory syncytial virus

In the following example describes the hybrid polypeptides of the respiratory syncytial virus (RSV) with enhanced pharmacokinetic properties. In addition, the presented data showing that hybrid RSV polypeptides are potent inhibitors of RSV infection.

8.1. Materials and methods

8.1.1. Synthesis and purification of peptides

RSV peptides are synthesized using standard methods Fast Mos chemistry. Basically, unless explicitly noted, the peptides contain amidarone carboxyl ends and acetylated amino ends. Cleaning avodat using HPLC with reversed phase.

8.1.2. Test for respiratory syncytial virus lower belascoaran

All the necessary dilution of the peptides are doing on a clean, sterile 96-well TC tablet. Prepare a total of eleven dilutions of each peptide and leave one control wells without peptide. The range of final concentrations of peptide starts with 50 μg/ml or 100 μg/ml, only eleven dilutions in two repetitions. RSV receive at a concentration of 100 CFU/well in 100 ml of 3% buffer EMEM, which was determined on the basis of known titer of RSV. Then the virus added to all wells.

Environment is withdrawn from the holes 96-well plate with cells Ner stage before the merge. The material of the tablet dilutions are transferred to the plates for cell culture, starting with number 1 and then transferred into a series of 12, 11 and so on until, until you made the transfer in all series. The tablets are then returned to the incubator for 48 hours.

Cells test to confirm the presence of syncytium in the control cells. Select the environment and to each well add about 50 μl of 0.25% crystal violet dye in methanol. Wells immediately washed with water to remove excess dye and leave to dry. Using preprofile magnifying glass to count the number of syncytium in each hole.

8.2. Results

Pharmacokinetic study the Oia, as can be seen from tiga-10V, hybrid peptides from RSV - T (AU-WQEWDEYDASISQVNEKINQALAYIREADELWA WF-NH2and T (AU-WQAWDEYDASISQVNEKINQALAYIREADELW AWF-NH2), containing the enhancer peptide sequence, demonstrated in these cases, significantly increased the half-life in comparison with crustal peptide T (AU-VYPSDEYDASISQVNEEINQALAYIRKADELLENV-NH2). For hybrid polypeptides T, T and T (AU-WQAWDEYDASISDVNEKINQALAYIREADELWEWF-NH2also shown significantly increased the half-life in comparison with crustal polypeptide C (Ac-DEYDASISQVNEKINQALAYIREADEL-NH2).

Hybrid polypeptides T, T and T, as well as polypeptides T and T examined for their ability to inhibit belascoaran in Ner cells under the action of RSV. As shown in figa and 11B, both the investigated hybrid polypeptide of RSV, as well as a core polypeptide T able to inhibit RSV infection. Unexpectedly it was found that the hybrid polypeptide C is also a powerful anti-RSV connection (Fig).

9. Example: hybrid polypeptides of luteinizing hormone

In the example below describes the hybrid proteins of the luteinizing hormone (LH) with reinforced pharmaco-kinetic properties. These hybrid peptides LH were synthesized and purified using the methods described above: the core peptide T (Ac-QHWSYGLRPG-NH2and hybrid polyp is ptid T (Ac-WQEWEQKIQHWSYGLRPGWASLWEWF-NH 2), which includes the amino acid sequence of the crustal polypeptide C, combined with enhancer polypeptides by amino - and carboxy-ends. As shown in figa and 12V hybrid peptide T has significantly increased the half-life in comparison with crustal peptide T that does not contain enhancer peptide sequences.

10. Example: pharmacology hybrid polypeptide T

T depicted on Fig, is a hybrid polypeptide comprising enhancer peptide sequence connected with crustal polypeptide obtained from a mixture of viral sequences. As was shown above, in the example of section 7, the hybrid polypeptide T shows enhanced pharmacokinetic properties and activity in vitro and in vivo against HIV-1. In the example below, the described pharmacological properties T on the models such as rodents and primates.

10.1. Materials and methods

10.1.1. A single dose of rodents

T enter albino rats sprag-Share (Sprague-Dawley) as a single dose by continuous subcutaneous infusion (CRP), subcutaneous injection (p/C) or intravenous injection (in/in). Each experimental group consists of nine rats of each sex per group. Groups of animals injected with sterile preparations of medicinal substance in the mass T in dose is 0,5, of 2.0 or 6.5 mg/kg by CRP. One group serving as control, gets 50 mm carbonate-bicarbonate, pH 8.5. Peptides injected within 12 hours through the polyvinyl chloride/polyethylene catheter, implanted subcutaneously surgically in the back of the neck. Two groups are in the form of subcutaneous injections in intracapsularly plot a single dose T equal to 1.2 or 1.5 mg/kg other Two groups are in the form of intravenous injection single dose T equal to 1.5, or 5 mg/kg the Actual number T in milligrams calculated on the basis of the content of the peptide defined for each input of a batch.

The parameters that are essential for the analysis include the observation of animals in the cage (twice daily records of deaths and diseases), clinical examination, laboratory tests, determination of body weight and conducting the necropsy. Blood samples obtained using the method of random sampling within 12-hour period from three rats of each sex from each group in each of the following time points: 0,5, 1, 2, 4, 6, 8, 10 and 12 hours after dose. Analyze samples using PcAb ECLIA test (Blackburn, G. et al., 1991, Clin. Chem. 37: 1534-1539; Deaver, D., 1995, Nature 377: 758).

For conducting pharmacokinetic analysis T in the blood and the lymphatic system of rats T prepared in the form of a sterile solution bicarbonate buffer and enter it in the form of a single dose of intravenous bolus injection in the lateral tail vein at a dose of 20 mg/kg Blood is taken with a permanent catheter placed in the jugular vein of the animal. Samples collected immediately after a dose and after 5, 15 and 30 minutes, and 1, 2, 4 and 6 hours after drug administration. For the analysis of lymphatic fluid samples taken immediately before the dose and every 20 minutes for six hours after a dose. Lymphatic fluid is collected by means of a catheter placed directly into the thoracic duct, as described previously (Kirkpatrick and Silver, 1970, The Journal of Surgical Research 10: 147-158). Concentration C in plasma and lymphatic fluid is determined using standard T on competitive IPTF method (Hamilton, G. 1991, R in "Immunochemistry of Solid-Phase Immunoassay", Butler, J., ed., CRC Press, Boston).

10.1.2. The introduction of a single dose of primates

Sterile drugs, bulk drug substances T administered to cynomolgus monkeys as a single dose by subcutaneous (s/C), intramuscular (I/m) or intravenous (IV) injection. In sequential cross-experiences of one group of animals, including two animals of each sex, administered a single dose of T bolus/(0.8 mg/kg)/m (0.8 mg/kg) or p/(of 0.4, 0.8 and 1.6 mg/kg) injection. Between doses make a break for cleansing lasting at least three days. Liofilizovannye T restore in sterile phosphate-buffered Rast is the PR (s) at pH 7.4 just before the introduction of the dose. The actual number of the investigated product in milligrams calculated based on the content of a peptide known to enter the party.

The parameters that are essential for the analysis include the observation of animals in the cage, physical examination, and consideration of body weight. In the course of/in the phase I study conducted sampling of blood in heparinized tubes at the following time points: immediately after a dose and through 0,25, 0,5, 1,5, 3, 6, 12 and 24 hours after dose. During I/m and p/phases of the study, carried out the sampling of blood from each animal into heparinized tubes at the following time points: through 0,5, 1, 2, 3, 6, 12 and 24 hours after dose. Plasma samples prepared within one hour in the selection process and quickly frozen in liquid nitrogen. Analysis of samples is carried out with the use of PcAb ECLIA test (Blackburn G. et al., 1991, Clin. Chem. 37: 1534-1539; Deaver, D., 1995, Nature 377: 758).

10.1.3. Combining pharmacokinetic studies

Six male cynomolgus monkeys are distributed randomly in three groups of two animals per group. All doses T administered by subcutaneous bolus injection. The study is divided into two sessions. In session 1 the animals in groups 1, 2 and 3 enter a sterile preparation of bulk drug substances T (for example, bulk T dissolved in carbonate-bicarbonate buffer, pH 8.5) twice a day in those is a group of four consecutive days (days research 1-4) at doses of 0.2, 0.6 and 2.0 mg/kg/dose, respectively. Next is a ten-day period of purification of the body, dividing the session 1 and session 2. During session 2 animals in groups 1, 2 and 3 enter the sterile drug product T (i.e. an aqueous solution of pH 6.5 plus mannitol) twice a day for four consecutive days (days of the study 15-18) at doses of 0.2, 0.6 and 2.0 mg/kg/dose, respectively.

Blood samples for pharmacokinetic analysis selected in the 1st and the 15th days of the study to assess the pharmacokinetic parameters of the effect of a single dose and at 4 and 18 days of the study to assess the pharmacokinetic parameters of plasma in the steady state. Samples taken at the following time points: immediately after a dose and 0.5, 1,5, 3, 6, 8 and 12 hours after dose. Animals observed during the 1st and 2nd sessions to identify clinically significant signs and changes in body weight.

10.2. Results

10.2.1. Pharmacokinetics T when a rat

For the initial assessment of pharmacokinetic parameters from plasma and the nature of the distribution in the body T use the model in rats. In all dosing groups with the introduction of T not marked changes in body weight of animals, abnormalities detected during physical examination, hematological parameters, clinical chemical analysis and when the macro is picscom examination.

Rats treated with T with CRPS reach steady state at peptide concentrations in plasma after about four hours after injection. And plasma concentration, which is characteristic for the stationary condition (KPSS), and the calculated value of the peak area on the graph according to the plasma concentration (AUC) is proportionate to the dose, indicating that T obeys linear pharmacokinetics in the studied dose range from 0.5 to 6.5 mg/kg Curves calculated pharmacokinetic parameters and values of the concentrations in plasma from the time when the way of the CRP are presented in Table 9 and figa respectively.

Table 9
Group dosing
0.5 mg/kg2.0 mg/kgthe 6.5 mg/kg
KPSS(ág/ml)0,802,8010,9
AUC(0-12 hour)(ug·h/ml)7,9925,9120

In the case of the introduction of T bolus/injection is linear character of the dependence of the pharmacokinetics of the dose range studied doses. In contrast, with the introduction of T s/C injection is not observed dependent on the capabilities of the dose in the studied dose range. Curves calculated pharmacokinetic parameters and values of the concentrations in plasma from the time when s/on/in the introduction are presented in Table 10 and figv respectively.

Table 10
Group dosing/introduction
(p/K)(in/in)
1.2 mg/kg15 mg/kg1.5 mg/kg5.0 mg/kg
t1/2, ConECs.(h)2,022,002,46to 1.86
tmax(h)1,091,88--
Withmax(ág/ml)6,37a 21.515,746,3
AUC(0-12 hour)(ug·h/ml)27,010745,6118
AUC(0-∞)(ug·h/ml)27,611047,1120

Bioavailability T, administered to rats subcutaneously, determine in comparison with its value when the/in the introduction. The results are shown below in Table 11. Low dose (1.2 mg/kg) T demonstrates percutaneously relative bydocument is (F R), equal to 73%. The relative bioavailability of 30% in the case when a high dose (15 mg/kg) administration T achieved concentration, most of the concentration that inhibits 90% (IC) the infectivity of HIV-1 within 12 hours of testing, all studied doses.

Table 11
Route of administrationDose (mg/kg)AUC(0-∞)(ug·h/ml)Normalized AUC(0-∞)(ug·h/ml)FR(%)
Low dose
n/a1/227,634,5(a)73
in/at1,547,1--
High dose
n/a1511036,5(b)30
in/at5120--
(a)Normalized dose 1.2 mg/kg up to a dose of 1.5 mg/kg by multiplying AUC(0-∞)1.25.
(b)Normalized to the dose of 15 mg/kg to dose mg/kg division AUC (0-∞)3.

Kinetic data for concentrations C in blood plasma and lymph shown in figs further reflected in Table 12. T quickly penetrates into the lymphatic system and equilibrated with plasma level of drug substances for approximately one hour after injection. After reaching equilibrium between the two systems levels of drug substance in plasma and lymph become comparable for the period of up to three hours after a dose at four of the five animals. One animal has a lower concentration T in the lymph than other animals, but the profile removal of lymph in the specified animal does not differ from that seen in other animals in the same group. Comparison of the half-life of excretion phase (t1/2from plasma and lymph suggests that moving T between these two systems is controlled by the diffusion process. Three hours later develops a second, more rapid phase-out of the lymphatic system. This difference can be determined by mechanism of action (for example, in connection with redistribution or accelerated destruction of the peptide in the lymph) or other factors. Concentration C in the lymph fluid through six hours after the injection is higher than the value IR90to maintain viral infectivity usual the laboratory strains and primary clinical isolates of HIV-1.

We also evaluate the penetration T in the cerebrospinal fluid (CSF). Concentration C were below the limit of detection (LOD: 2.0 µg C/ml CSF) in all measured time points, indicating that T does not penetrate into the Central nervous system after administration of a single dose.

Table 12
T
PlasmaLymph
t1/2excretion (h)2,6±0,411,3±0,27
Withmax(ág/ml)291133(a)/l55(b)
AUC(0-6 hour)(ug·h/ml)505348(a)/411(b)
AUC(0-∞)mcg·h/ml)598390(a)/449(b)
Cl(ml/h)7,811,5
(a)The calculated average values, including one animal (rat No. 1), who observed lower concentrations in the lymph, but similar kinetic profile when compared with other animals in the group.
(b)The calculated average values, which exclude data for rats No. 1.

10.2.2. Farmacocinetica with the introduction of the apes

Primate models used to assess the level of dose and various pharmacokinetic parameters with a parenteral T. Concentration T plasma, higher than 6,0 ág/ml are achieved when all routes of administration, and quantifiable levels (i.e. levels, large 0.5 μg/ml) are detected by 24 hours after p/and/in introduction. The values of half-life t1/2excretion comparable for all routes of administration (5.4 hours 4.8 hours and 5.6 hours for/p/and/m injection, respectively). All time points during a 24-hour period of sampling observed plasma concentration T exceeding values IR90for laboratory strains and clinical isolates of HIV-1.

Comparison of data obtained in the case of parenteral administration of 0.8 mg/kg T using all routes of administration (p/K/and/m) presented on figa. On FIGU shows the comparison of data obtained by s/C injection of three different doses of T (0.4 mg/kg, 0.8 mg/kg and 1.6 mg/kg). On the insert shown in figv shown a graph of the calculated concentration and the AUC of the administered dose.

T demonstrates linear pharmacokinetics in the body of cynomolgus monkeys when s/introduction to the range of the investigated doses, indicating the absence in the specified range of the saturation mechanism or mechanisms of clearance. Brief summarizes the E. pharmacokinetic data parenteral T to cynomolgus monkeys given below in Table 13. Comparison of plasma AUC values shows that, relative to the intravenous mode of administration, the bioavailability T approximately 64% with the introduction of intramuscular injection and 92% with the introduction of subcutaneous injection.

Table 13
Route of administration (dose, mg/kg)
P/C (0,4)P/C (0,8)P/C (1,6)V/m (0,8)In/in (0,8)
t1/2konecne (h)6,23±0,52a 4.83±0,485,55±0,925,57±0,245,35±0,95
tmax(UNEF)3,97±1,184,58±1,454,72±1,812,32±0,43-
Withmax(ág/ml)3,17±0,096,85+1,0113,3±2,556,37±1,6926,7±0,25
AUC(0-24 hours)(ug·h/ml)37,5±6,68,12±11,4168±34,0of 56.4±12,387,4+25,0
AUC(0-∞)(ug·h/ml)40,9±8,285,3±13,6181±44,059,5±13,192,5±25,0
fR (%)-92,3-64,4-

10.2.3. Combining pharmacokinetic studies

Combining pharmacokinetic studies conducted to compare the pharmacokinetic profiles in the plasma bulk drug substances T used in the above-described non-clinical trials, drug product T, which is specific to a subject or patient, for example, for the treatment of HIV infection. The study was conducted in parallel groups, in the form of unidirectional cross-comparison of the three values of bulk doses of medicinal substances T and three values of doses prepared medicinal product. Pharmacokinetics in plasma assessed after administration of a single dose and after reaching steady state.

After the introduction T subcutaneous injection in all dosing groups indicates the presence of a detectable when measuring levels of the peptide. Curves concentrations in plasma were in General parallel within all groups dosing after receiving initial doses (days 1 and 15) and in the steady state (days 4 and 18) for bulk drug substances T and medicinal product T. In addition, the values of AUC(0-12 hour)changed in direct proportion to the depending on the level of dose of each form of the drug. The calculated values of the AUC(0-12 hour)for drug product varies from 43% to 80% of the AUC(0-12 hour)calculated for medicinal substance after administration of a single dose, and from 36% to 71% in the steady state.

T in the form of bulk drug substance and drug product demonstrates similar pharmacokinetic profiles in cynomolgus monkeys after injection of a bolus dose of the investigated level and volume. The results of the direct comparison of the curves of the dependence of concentration on time in the plasma in this study and the shapes of the curves in the previous study in cynomolgus monkeys suggest the presence of a depot effect in the case of the introduction of T subcutaneous injection. This assumption follows from the fact that increasing the time during which the maximum plasma concentration (tmax) and the values of t1/2.

The results indicate that the form of bulk drug substances used in pharmaceutical program gives comparable values of AUC and other pharmacokinetic parameters with those that are observed after the introduction of the finished product. The above observations show that the introduction T clinically leads to the achievement of a comprehensive impact is of major T on the entire body of the patient.

11. Example: allocation of new crustal polypeptides with antiviral activity against HIV-1 isolate resistant to T

In one specific, but non-limiting example of the present description discusses getting modified crustal polypeptide that exhibits antiviral activity against HIV strains that are resistant to remotefilename, "parent" korovou the peptide.

Peptide T specified in Table 2, is a peptide derived from the plot Dr protein of HIV-1, also known as HR2. In studies of variants of HIV-1 resistant T, selecting and sequencing a nucleic acid that encodes HR2 area of drug-resistant variants Dr protein identified mutation, which leads to a single mutation within the specified plot: the replacement of the asparagine residue (N) for lysine (K).

Based on this result was synthesized polypeptide used in the present description as DP397, which contains the amino acid sequence T in which you have entered the above mutation N K. Peptides T and DP397 shown below where you can see that between them there is a difference in a single amino acid in bold:

T: WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL

DP397: WMEWDREINBy KytSLIHSLIEESQNQQEKNEQELL

It should be noted that the distinction between T and DP397 gets in on entially site N-glycosylation sites (underlined). Thus, the mutation in Dr at T-resistant strains removes the specified potential site of N-glycosylation.

A core polypeptide DP397 exhibits antiviral activity against variants of HIV-1 that are resistant to peptide T. In particular, the peptide DP397 shows markedly increased antiviral activity against four variants of HIV-1, according to test infectivity for Magi-CCR-5, previously described in section 7.1.7. In addition, it was also shown in several experiments that the peptide DP397 has increased antiviral activity against these strains in comparison with peptide T.

On figa-D shows the number of infected cells when exposed to T-resistant variants as a function of the concentration of peptide for T, DP397 and T. Specifically, figa In the data of experiments using T-resistant strains of HIV-1, referred to as RF-649 and DH012-649 respectively. These strains were obtained from isolates of HIV-1RFand HIV-1DHO12respectively, which have been passaged in cell culture in the presence of T order products T-resistant variants.

On figs-D show experimental data obtained using genetic engineering methods T-resistant strains of HIV-1, called 3'ETVQQQ and SIM 649 respectively. Strain 3'ETVQQQ obtained from a clone In The H-1 LAIsubjected to molecular mutations, in which it contains the amino acid sequence ETVQQQ instead GIVQQQ in the HR1 domain Dr protein. HR1 is a plot Dr protein of HIV-1, which are associated HR2 domain and C peptide. Strain SIM-649 was obtained from a clone of HIV-1LAIsubjected to molecular mutations, in which it contains the amino acid sequence SIM instead of GIV in the HR1 domain Dr protein, and subsequently passaged in cell culture in the presence of T order products T-resistant variants.

Peptide DP397 demonstrates significantly enhanced inhibition of HIV-1 infection in comparison with T for all four tested strains. In addition, the peptide DP397 demonstrates enhanced inhibition of HIV-1 infection in comparison with T for RF-649 strain (figa) and, at higher concentrations, for DH012-649 strain (pigv).

The present invention is not limited to the above description of specific variants of its implementation, which are given merely to illustrate certain aspects of the invention, and functionally equivalent methods and components are within the scope of the present invention. In fact, for specialists in this area apparent various modifications that can be introduced in the invention based on the above description and accompanying ri is uncov. Such modifications also fall within the scope of the invention and the accompanying claims.

1. A way to enhance the pharmacokinetic properties of the crustal polypeptide comprising a binding enhancer peptide sequence with crustal polypeptide with obtaining a hybrid polypeptide, where the specified enhancer peptide sequence includes WXXWXXXI, WXXWXXX, WXXWXX, WXXWX, WXXW, WXXXWXWX, XXXWXWX, XXWXWX, XWXWX, WXWX, WXXXWXW, WXXXWX, WXXXW, IXXXWXXW, XXXWXXW, XXWXXW, XWXXW, XWXWXXXW, XWXWXXX, XWXWXX, XWXWX, XWXW, WXWXXXW or XWXXXW, where a core polypeptide comprises TALLEQAQIQQEKNEYELQKLDK and where at least one amino acid residue in a hybrid polypeptide anywhereman with the polyol, so that when introduced into a living system specified hybrid polypeptide exhibits antiviral activity and enhanced pharmacokinetic properties in comparison with the activity and properties, which shows a core polypeptide.

2. Hybrid polypeptide containing the enhancer peptide sequence associated with crustal polypeptide, where the enhancer peptide sequence derived from a consensus sequence of a protein gp-41 membrane of HIV-1, HIV-2 or SIV containing WXXWXXXI, WXXWXXX, WXXWXX, WXXWX, WXXW, WXXXWXWX, XXXWXWX, XXWXWX, XWXWX, WXWX, WXXXWXW, WXXXWX, WXXXW, IXXXWXXW, XXXWXXW, XXWXXW, XWXXW, XWXWXXXW, XWXWXXX, XWXWXX, XWXWX, XWXW, WXWXXXW or XWXXXW, and where the crust polypeptide sequence comprises the following amino acid PEFC is the sequence:

YTSLIHSLIEESQNQQEKNEQELLELDK;LEENITALLEEAQIQQEKNMYELQLNS;
LEANISQSLEQAQIQQEKNMYELQKLNS;NNYTSLIHSLIEESQNQQEKNEQELLEL;
DFLEENITALLEEAQIQQEKNMYELKL;RYLEANISQSLEQAQIQQEKNMYELQKL;
RYLEANITALLEQAQIQQENEYELQKL;
TALLEQAQIQQEKNEYELQKLDK;TALLEQAQIQQEKNEYELQKLDE
TALLEQAQIQQEKNEYELQKLIE;TALLEQAQIQQEKIEYELQKLDK;
TALLEQAQIQQEKIEYELQKLDE;TALLEQAQIQQEKIEYELQKLIE;
TALLEQAQIQQEKIEYELQKLE;TALLEQAQIQQEKIEYELQKLAK;
TALLEQAQIQQEKIEYELQKLAE;TALLEQAQIQQEKAEYELQKLE;
TALLEQAQIQQEKNEYELQKLE;TALLEQAQIQQEKGEYELQKLE;
TALLEQAQIQQEKAEYELQKLAK;TALLEQAQIQQEKNEYELQKLAK;
TALLEQAQIQQEKGEYELQKLAK;TALLEQAQIQQEKAEYELQKLAE;
TALLEQAQIQQEKNEYELQKLAE;TALLEQAQIQQEKGEYELQKLAE;
DEYDASISQVNEKINQALAYIREADEL;DEYDASISQVNEEINQALAYIRKADEL;
DEFDESISQVNEKIEESLAFIRKSDELL;or
DEFDESISQVNEKIEESLAFIRKSDEL

and where at least one amino acid residue of the hybrid polypeptide anywhereman with the polyol.

3. A hybrid polypeptide according to claim 2, in which a core polypeptide contains TALLEQAQIQQEKNEYELQKLDK.

4. A hybrid polypeptide according to claim 2, in which a core polypeptide contains NNYTSLIHSLIEESQNQQEKNEQELLEL.

Hibiny polypeptide according to claim 2, in which a core polypeptide contains YTSLIHSLIEESQNQQEKNEQELLELDK.

6. A hybrid polypeptide according to claim 2, 3, 4 or 5, in which the enhancer peptide sequence is associated with aminocom.com crustal polypeptide.

7. A hybrid polypeptide according to claim 6, additionally containing the enhancer peptide sequence associated with the carboxyl end of the crustal polypeptide.

8. A hybrid polypeptide according to claim 2 to 6 or 7, in which the enhancer peptide sequence contains WQEWEQKI or WASLWEWF.

9. The hybrid polypeptide under item 8, containing the amino acid sequence WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF.

10. A hybrid polypeptide according to claim 2, in which the enhancer peptide sequence is linked to the carboxyl end of the crustal polypeptide.

11. The hybrid polypeptide under item 10, in which a core polypeptide contains TALLEQAQIQQEKNEYELQKLDK.

12. The hybrid polypeptide under item 10, in which a core polypeptide contains NNYTSLIHSLIEESQNQQEKNEQELLEL.

13. A hybrid polypeptide according to any one of claim 2 to 8, 10-12, optionally containing aminobenzene acetyl group and carboxykinase aminogroup.

14. A hybrid polypeptide according to claim 9, further containing aminobenzene acetyl group and carboxykinase aminogroup.

15. A hybrid polypeptide according to any one of claim 2 to 8, 10-12, where the core polypeptide is a therapeutic agent.

16. Hybrid polypep the ID according to claim 9, where the core polypeptide is a therapeutic agent.

17. A hybrid polypeptide according to any one of claim 2 to 8, 10-12, where the polyol is a poly(propylene glycol), polyethylene-polypropyleneglycol or poly(ethylene glycol).

18. A hybrid polypeptide according to 17, where the polyol is a linear-chain polyol.

19. A hybrid polypeptide according to 17, where the polyol is a branched polyol.

20. A hybrid polypeptide according to claim 9, where the polyol is a poly(propylene glycol), polyethylene-polypropyleneglycol or poly(ethylene glycol).

21. The polypeptide according to claim 20, where the polyol is a linear-chain polyol.

22. The polypeptide according to claim 20, where the polyol is a branched polyol.

23. The polypeptide containing TALLEQAQIQQEKNEYELQKLDK, where at least one amino acid residue of the polypeptide anywhereman with the polyol.

24. The polypeptide according to item 23, further containing aminobenzene acetyl group and carboxykinase aminogroup.

25. The polypeptide according to item 23, where the core polypeptide is a therapeutic agent.

26. The polypeptide according to item 23, where the polyol is a poly(propylene glycol), polyethylene-polypropyleneglycol or poly(ethylene glycol).

27. The polypeptide according p, where the polyol is a linear-chain polyol.

28. The polypeptide according p, where polio is a branched polyol.

29. Pharmaceutical composition for the inhibition of HIV-1 or HIV-2, containing polypeptide TALLEQAQIQQEKNEYELQKLDK or WQEWEQMTALLEQAQIQQEKNEYELQKLDKWASLWEWF, where at least one amino acid residue in the polypeptide anywhereman with a polyol, and a pharmaceutically acceptable carrier.

30. The pharmaceutical composition according to clause 29, additionally containing aminobenzene acetyl group and carboxykinase aminogroup.

31. The composition according to clause 29, where the core polypeptide is a therapeutic agent.

32. The composition according to clause 29, where the polyol is a poly(propylene glycol), polyethylene-polypropyleneglycol or poly(ethylene glycol).

33. The composition according to p, where the polyol is a linear-chain polyol.

34. The composition according to p, where the polyol is a branched polyol.

35. Hybrid polypeptide containing the enhancer peptide sequence associated with crustal polypeptide, where the specified enhancer peptide sequence includes WQEWEQKI or WASLWEWF and where at least one amino acid residue in a hybrid polypeptide anywhereman with the polyol.

36. A hybrid polypeptide according p, in which the enhancer peptide sequence is associated with aminocom.com crustal polypeptide.

37. A hybrid polypeptide according p, optionally containing enhancer peptide the second sequence, associated with the carboxyl end of the crustal polypeptide.

38. A hybrid polypeptide according p, in which the enhancer peptide sequence is linked to the carboxyl end of the crustal polypeptide.

39. A hybrid polypeptide according p, in which the enhancer peptide sequence includes WQEWEQKI.

40. A hybrid polypeptide according p, in which the enhancer peptide sequence includes WASLWEWF.

41. A hybrid polypeptide according to any one of p-40, optionally containing aminobenzene acetyl group and carboxykinase aminogroup.

42. A hybrid polypeptide according to any one of p-40, in which the core polypeptide is a therapeutic agent.

43. A hybrid polypeptide according to any one of p-40, in which the core polypeptide is a bioactive peptide, exhibiting antiviral activity, growth factor, cytokine or hormone.

44. A hybrid polypeptide according to any one of p-40, where the polyol is a poly(propylene glycol), polyethylene-polypropyleneglycol or poly(ethylene glycol).

45. A hybrid polypeptide according to item 44, where the polyol is a linear-chain polyol.

46. A hybrid polypeptide according to item 44, where the polyol is a branched polyol.

47. The polypeptide containing the amino acid sequence VYPSDEYDASISQVNEEINQALAYIRKADELLENV, where less than the least one amino acid residue in the polypeptide anywhereman with the polyol.

48. The polypeptide according p, optionally containing aminobenzene acetyl group and carboxykinase aminogroup.

49. The polypeptide according p, in which the core polypeptide is a therapeutic agent.

50. The polypeptide according p, where the polyol is a poly(propylene glycol), polyethylene-polypropyleneglycol or poly(ethylene glycol).

51. The polypeptide according to item 50, where the polyol is a linear-chain polyol.

52. The polypeptide according to item 50, where the polyol is a branched polyol.



 

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Oligopeptides // 2260597

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