Hybrid polypeptides with improved pharmacokinetic properties
The present invention relates to sequences of peptide-amplifier originating from different sequences of the envelope protein of the retrovirus (gp41), which improves the pharmacokinetic properties of any crustal polypeptide to which they are attached, and based on the detection of the fact that the hybrid polypeptides containing sequences of peptide-amplifier attached to korovou the polypeptide, have improved pharmacokinetic properties, such as a longer half-life. The present invention also relates to a method for improving the pharmacokinetic properties of any crustal polypeptide by joining these two sequences of peptide-amplifier to the specified korovou the polypeptide. Core polypeptides, used to implement the present invention may be any pharmacologically acceptable peptides that can be used, for example, as a therapeutic or prophylactic agent. The advantage of the invention is to improve the effectiveness of biologically active peptides in the bloodstream. 3 N. and 35 C.p. f-crystals, 13 tab., 26 Il.
Nostoapuvalineet envelope protein of a retrovirus (gp41) and improving the pharmacokinetic properties of any crustal polypeptide, with which they are associated. The present invention is based in part on the discovery of the fact that the hybrid polypeptides containing sequences of peptide-amplifier attached to korovou the polypeptide, have improved pharmacokinetic properties, such as a longer half-life. The present invention also relates to new antimutagennym and/or antiviral peptides, including peptides containing the above-mentioned sequence of the peptide-amplifier, and to methods of using such peptides. The present invention also relates to methods of improving the pharmacokinetic properties of any crustal polypeptide by joining this korovou the specified polypeptide sequences of the peptide-amplifier. The crust polypeptides used in the present invention, can include any pharmacologically acceptable peptides that can be used, for example, as a therapeutic or prophylactic agent. In a non-limiting example illustrating a variant implementation of the present invention, it has been demonstrated that the hybrid polypeptide containing, for example, a core polypeptide of HIV-joined pole, the sequence of peptide-amplifier of the present invention were attached to korovou the polypeptide respiratory syncytial virus (RSV) and korovou the polypeptide receptor, luteinizing hormone (LH-RH). In each case it was found that the hybrid polypeptide has improved pharmacokinetic properties, and the hybrid polypeptide based on the RSV has significant activity against RSV.
2. Background of invention
Polypeptide products are widely used as therapeutic and/or prophylactic agents for the prevention and treatment of diseases. Many of these polypeptide capable of regulating a biochemical or physiological process that leads either to the prevention of disease, or to reduce the symptoms associated with these disease. So, for example, polypeptides such as viral or bacterial polypeptides, can be successfully used as vaccines for the prevention of pathological conditions. In addition, peptides can be successfully used as therapeutic agents for the treatment of symptoms. Such peptides are proteins of different categories, such as, for example, hormones, thebiological and therapeutic effect at the site of the target, they must be present in these sites in appropriate concentrations. In addition, should, in the main, retained their structural integrity. Therefore, the composition of the polypeptides for therapeutic use as medicines depends on the chemical nature and characteristics of these polypeptides, such as size and structure, conformational requirements, and often difficult to predict stability and solubility parameters. Pharmacokinetic properties of any specific therapeutic peptide-dependent bioavailability, distribution and excretion of the indicated peptide.
Since many biologically active substances, such as peptides and proteins are rapidly destroyed in the body, to increase the efficiency of these peptides and to minimize the occurrence of dangerous side effects and their severity at the present time, it is imperative to develop an effective system to maintain a stable concentration of peptide in the bloodstream.
3. Brief description of the invention
The present invention mainly relates to sequences of peptide-amplifier, a source originating from different sequences of the envelope protein Retrovir they are attached. The present invention is based on the discovery of the fact that when the described sequence of peptide-amplifier attached to any korovou the polypeptide, the resulting hybrid polypeptide has improved pharmacokinetic properties, including, for example, a longer half-life and lower the rate of its removal from the bloodstream, compared to crustal polypeptide, taken separately. The present invention also relates to these hybrid polypeptides and crustal polypeptides, as well as to new peptides, which have antipathogenic activity, antiviral activity and/or the ability to modulate intracellular processes associated with piperlime peptide structures. Such peptides are peptides that contain sequences of peptide-amplifier.
The crust polypeptides can include any peptides that may be introduced into a living system, such as any peptides that are able to function as therapeutic, prophylactic or imaging agents used to treat or prevent disease, or which can be used in diagnostic is s growth hormones, cytokines, angiogenic growth factors, extracellular polypeptides matrix, receptor ligands, agonists, antagonists or inverse agonists, peptide agents for delivery, such as visualizing agents or cytotoxic agents for delivery, or polypeptides that have antipathogenic and/or antiviral activity, as well as peptides or polypeptides that function as antigens or immunogenic, including, for example, viral and bacterial polypeptides.
The present invention also relates to methods of improving the pharmacokinetic properties of any measles-containing polypeptide by attaching the crustal polypeptide to the sequence of the peptide-amplifier with the formation of the hybrid polypeptides.
The present invention also relates to methods of using the peptides described herein, including hybrid polypeptides containing sequences of peptide-amplifier. For example, the methods of the present invention are methods of attenuating or inhibiting a viral infection, such as HIV-1, HIV-2, RSV, measles, influenza, parainfluenza, Epstein-Barr and hepatitis, and/or events induced by virus merge cells. These sequences stumps in vitro or ex-vivo crustal polypeptide, attached sequence peptide-amplifier, for example the sequence of the peptide amplifier can increase the half-life of attached core polypeptides in samples of cell cultures, cells or tissues.
The present invention is demonstrated in the examples, where it is shown that the hybrid polypeptides containing a core polypeptide of HIV, attached to the peptide sequences amplifier have substantially improved pharmacokinetic properties and act as a strong recitations inhibitors of HIV-1, HIV-2 and SIV infection. The present invention also demonstrated in the examples, where it is shown that the hybrid polypeptides containing a core RSV polypeptide or polypeptide of luteinizing hormone, have significantly improved pharmacokinetic properties. In addition, the hybrid polypeptide based on the RSV has significant activity against RSV.
Peptides, polypeptides and proteins identified in the present description as organic compounds containing two or more amino acids, covalently linked, for example, the peptide amide bonds. Peptides, polypeptides and proteins can cacioppi, described in this application. Therefore, the terms "peptide", "polypeptide" and "protein" are used here interchangeably.
The peptide sequence defined in the present description, represented by single letter symbols used to denote the following amino acid residues:
D (aspartic acid)
E (glutamic acid)
X (any amino acid)
"The sequence of the peptide-amplifier" is defined as peptides 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 and can be any amino acid, W means tryptophan, and I is isoleucine. As described below, the sequence of the peptide-amplifier of the present invention is also a peptide sequence that more or less similar to the consensus amino acid posledovatelnostei pharmacokinetic properties of the crustal peptide, with which it is associated, compared with the pharmacokinetic properties of the specified crustal polypeptide, taken separately.
Used herein, the term "core polypeptide" refers to any polypeptide that can be introduced into a living system and, thus, is a biologically active molecule, for example, any polypeptide that can act as a pharmacologically effective peptide for the treatment or prevention of disease.
Used herein, the term "hybrid polypeptide" means a polypeptide containing amino-, carboxy - or amino - and carboxykinase sequence of peptide-amplifier and a core polypeptide. Typically, the sequence of the peptide amplifier connected directly to korovou the polypeptide. It should be noted that the peptide-amplifier can also be attached to the intermediate amino acid sequence, localized between the sequence of the peptide-amplifier and crustal peptide.
Used herein, the terms "antipathogenic" and "directed against the fusion of membranes" refers to the ability of the peptide to inhibit or reduce the number of events of a merger between two or more structures, for example between cell membranes or VI is in the absence of this peptide.
Used herein, the term "virus" refers to the ability of the peptide to inhibit viral infection of cells, for example, by merging cells or infection free viral particle. This infection may include fusion of the membranes, as in the case of viruses, shell, or other event of a merger occurring between viral structure and cell structure, for example the fusion of viral pilej and bacterial membrane during conjugation bacteria.
4. Brief description of drawings
Fig.1. Hybrid polypeptides. Shows the sequence of the peptide amplifier, derived from the assumed N-terminal and C-terminal interactive areas and attached to korovou the polypeptide of the appropriate type. The conserved sequence of the peptide amplifier shaded. It should be noted that the sequence of the peptide-amplifier can be used either as N-terminal, C-terminal, or both N - and C-terminal additions. In addition, the sequence of the peptide-amplifier can be added to korovou the polypeptide in a direct or reverse orientation, individually or in all possible combinations, in order to improve the pharmacokinetic properties of this peptide.
Phi is) and represents the N-terminal interactive area, observed in all known sequences of isolates of HIV-1, HIV-2 and SIV. The final sequence of "WXXWXXXI" is a "consensus" sequence.
Fig.2B. Sequences of peptide-amplifier originating from different sequences of the envelope protein (gp41) and represents the C-terminal interactive region observed in all known sequences of isolates of HIV-1, HIV-2 and SIV. The final sequence of "WXXXWXWX" represents a consensus sequence.
Fig.3. Comparison of titers of HIV-1 in tissues of the mouse SCID-HuPBMC, infected with HIV-1 9320, as was measured by P24 levels in the analysis under cultivation with NHRVMS person. This figure illustrates the comparison of the in vivo inhibition of viral infection polypeptides T20 and T.
Fig.4A-4B. The pharmacokinetic profile T in plasma compared with the pharmacokinetic profile of the control of the crustal polypeptide C in CD rats after intravenous injection over a period of time up to 2 hours (Fig.4A) and 8 hours (Fig.4B). Polypeptide T is a core polypeptide, and the polypeptide T is a core polypeptide attached to the peptide sequences amplifier.
wow injection. Polypeptide T is a hybrid polypeptide containing a core polypeptide (T) attached to the peptide sequences amplifier. T20: n=4; T: n=3.
Fig.6. Comparison of activity and cytotoxicity T20/T directed against HIV-1/IIIb.
Fig.7. Direct linking T with gp41-design M41178.125I-T1249 was purified by HPLC to a maximum specific activity. Shows the binding to a state of saturation with M41178 (a hybrid protein with ectodomains gp41, not containing the amino acid sequence T20) immobilized on microtiter tablets at 0.5 mg/ml.
Fig.8. The curve of the Association/dissociation T depending on time. The results demonstrated that125I-T1249 and125I-T20 have identical affinity binding components of 1-2 nm. The initial rate of Association and dissociation for125I-T1249 is much lower than the speed for125I-T20. The dissociation of the bound radioligand was measured after joining unlabeled peptide to a final concentration of 10 μm in 1/10 of the total volume of analysis.
Fig.9. Comparison of binding T with M41178. Unlabeled T and T20 was titrated in the presence of odnogo peptide.
Fig.10A-10B. The pharmacokinetic profile RSV-hybrid polypeptides T (10A) and T (10B) in plasma compared with T in rats CD.
Fig.11A. Analysis to reduce the number of plaques. The hybrid polypeptide can inhibit RSV infection with IC50=2.6 mg/ml
Fig.11. Analysis to reduce the number of plaques showed the ability of RSV-hybrid polypeptides T, T and T to inhibit RSV infection.
Fig.12A and 12B. The pharmacokinetic profile of the hybrid polypeptide of luteinizing hormone T in plasma compared with T in male CD rats. Polypeptide T is a core polypeptide of luteinizing hormone, polypeptide C is a hybrid polypeptide containing a core polypeptide attached to the peptide sequences amplifier.
Fig.13. The sequence of the hybrid polypeptide originating from different crustal polypeptides. The crustal sequence of the polypeptide is shaded. Not shaded amino - and carboxykinase sequences are sequences of peptide-amplifier.
Fig.14A-Century Range of circular dichroism (CD) for T in solution (phosphate buffered saline, pH 7) separately (10 µm 1C, Fig.14A) and in combination with the cue range CD, predicted for "non-interactive" model, and the true CD spectra represented by the shaded circles (l).
Fig.15. The polyacrylamide gel electrophoresis demonstrated T-protection gp41-design M41178 from hydrolysis proteinases To; lane 1: marker-seed; lane 2: untreated M41178; lane 3: M41178, incubated with proteinase K; lane 4: untreated T; track 5: T, incubated with proteinase K; lane 6: M41178, incubated with T; track 7: incubation T and M41178 before adding proteinase K.
Fig.16A-C. Pharmacokinetics polypeptide T rats-albino Sprague-Dawley; Fig.16A: the pharmacokinetics of polypeptide T with the introduction of a single dose by continuous subcutaneous infusion; Fig.16B: the pharmacokinetics of polypeptide T introduced by subcutaneous injection (s.c.) or by intravenous injection (i.v.); Fig.16C: kinetic analysis T in lymph and plasma after intravenous administration.
Fig.17A-Century Pharmacokinetics polypeptide T at abacadabra monkeys; Fig.17A: the pharmacokinetics of polypeptide T plasma with the introduction of a single dose of 0.8 mg/kg T by subcutaneous (s.c.), intravenous (i.v the second injection T in three different doses (0.4 mg/kg, 0.8 mg/kg and 1.6 mg/kg).
5. Detailed description of the invention
Described herein peptide sequence called sequence peptide-amp were obtained from different sequences of the envelope protein (gp41) of retrovirus that can improve the pharmacokinetic properties of crustal polypeptides to which they are attached. Such sequences of peptide-amplifier can be used in ways to improve the pharmacokinetic properties of any crustal polypeptide by joining peptide sequences amplifier to korovou the polypeptide with the formation of the hybrid polypeptide with improved pharmacokinetic properties compared with crustal polypeptide, taken separately. The half-life of the crustal peptide that is attached (or connected) the sequence (or sequences) of peptide-amplifier, can also be increased in vitro. So, for example, attached sequence peptide-amplifier can increase the half-life of the crustal polypeptide with their presence in cell culture, tissue culture or in samples taken from the patient, such as cells, tissue or other samples.
The crust polypeptides of hybridiser any peptide, which can function as a therapeutic or preventive agent used for the treatment or prevention of disease, or as visualizing agent used to visualize structures in vivo.
In the present application is also described peptides, including peptides containing the sequence of the peptide-amplifier, which have antipathogenic and/or antiviral activity. In addition, the present application describes methods of using such peptides, including ways to reduce or inhibit viral infection and/or virus-induced fusion of cells.
5.1. Hybrid polypeptides
Hybrid polypeptides of the present invention contain at least one sequence of peptide-amplifier and a core polypeptide. Hybrid polypeptides of the present invention preferably contain at least two sequences of peptide-amplifier and a core polypeptide, with at least one peptide-amplifier is present in the specified hybrid polypeptide from aminobenzo towards korovou the polypeptide, and at least one sequence of peptide-amplifier is present in the specified hybrid polypeptide from carboxylic on otone sequence, source originating from different sequences of the envelope protein (gp41) of retrovirus, including sequences of HIV-1, HIV-2 and SIV, and their specific options or modifications described below. A core polypeptide can contain any peptide sequence, preferably any peptide sequence that can be introduced into a living system, including, for example, peptides, designed for use in therapeutic or prophylactic purposes or for the purposes of visualization.
Generally, the hybrid polypeptide has a length comprising from about 10 to about 500 amino acid residues, and preferably from about 10 to about 100 amino acid residues, and most preferably from about 10 to about 40 amino acids.
Without pretending to any particular theory, it can be noted that the structure of the protein shell is built, it is obvious, therefore, that the intended scope-spiral, localized in the C-terminal region of the protein, is associated with the scope latinboy lightning", localized in the N-terminal region of this protein. Comparative analysis of the primary N-terminal and C-terminal sequences of the peptide-amplifier with gp41 regions observed in all of the particular, identified the following "consensus" amino acid sequence representing the consensus sequence of the peptide-amplifier (listed below consensus sequences in forward and reverse orientation, as specified sequence of peptide-amplifier can be used either in direct or 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 tryptophan, and I is isoleucine. Consensus amino acid sequences in direct orientation shown in Fig.1 and 2.
Typically, the length of the sequence of peptide-amplifier is about 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, and preferably from about 4 to about 20 residues, more preferably from about 4 to about 10 residues, and most preferably from about 6 to about 8 residues.
In a preferred embodiment of the invention the sequence of the peptide-amplifier that can be used to improve the pharmacokinetic properties of the obtained hybrid polypeptid is 1. The most preferred peptide sequences amplifier are peptides containing the following amino acid sequence: "WQEWEQKI" and WASLWEWF.
As non-limiting examples, the following Table 1 lists the amino acid sequences that are included in the preferred options peptide sequences amplifier of the present invention. It should be noted that, although negativeone sequence presented in direct orientation, however, the scope of the present invention also include sequences in the reverse orientation. For example, although the following sequence WMEWDREI" peptide-amplifier is in direct orientation, however, this sequence in the reverse orientation, that is, "IERDWEMW", is also in the scope of the present invention.
In another preferred variant of the invention, the specific sequences of the present invention contain sequences of peptide-amplifier shown in Fig.2, 13 and in Table 1 and having conservative amino acid substitutions at one, two, or three positions, where these substitutions do not affect the ability of the sequence of peptide-amplifier at what peptidome.
Particularly preferably, such substitutions resulted in the formation of peptide-amplifier, which has in its sequence from the consensus sequences. Typically, such replacement is carried out in the amino acid residues corresponding to positions "X" shown in the consensus amino acid sequences presented above and in Fig.1 and 2. The term "conservative substitution" means the replacement of amino acid residues having similar charge and size, and/or the same hydrophobic/hydrophilic properties as the replaced amino acid residue. Such characteristics of the amino acids is well known to specialists.
The present invention also relates to sequences of peptide-amplifier containing the amino acid sequence shown in Fig.1, 2 and 13 and in Table 1, which in varying degrees are similar, but the sequence of the peptide amplifier has one or more amino acid additions (usually not more than about 15 amino acid residues), deletions (e.g., truncation at the amino - or [carboxy]-end) or non-conservative substitutions, which, however, do not affect the ability of the obtained peptide-amplifier to improve pharmacoki without such peptide sequences amplifier.
Add usually do not exceed about 15 amino acid residues, and may include adding about 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 peptide-amplifier does not exceed about 15 amino acid residues, more preferably, this number was not more than about ten amino acid residues, and most preferably, this number does not exceed about 5 amino acid residues.
The preferred divisions are deletions not exceeding in total about 3 amino acid residue (either consecutive or not consecutive residues), more preferred are deletions from 2 amino acids, and most preferred are deletions of one amino acid residue. Usually deletions of amino acid residues correspond to residues "X" consensus peptide sequences amplifier.
The sequence of peptide-amplifier of the present invention also contain a specific sequence of peptide-amplifier shown in Fig.2, 13 and in Table 1 and having one, two or three non-conservative amino acid substitution, exchange means replacing the amino acid residues, with another charge, size, and/or hydrophobic/hydrophilic properties compared to the replaced amino acid residue. Such properties of amino acids are well known to specialists.
In addition, replacement of amino acids preferably should not be limited to, and in some embodiments of the invention preferably are not limited to the genetically encoded amino acids. Indeed, these peptides may contain non-coding genetically amino acids. Thus, in addition to natural genetically encoded amino acids, amino acid residues in these peptides can be replaced by non-coding nature of amino acids and synthetic amino acids.
Some of the most commonly encountered amino acids, which can be used as a suitable substitutions include, but are not limited to,-alanine (-l) and other omega-amino acids such as 3-aminopropionic acid, 2,3-diaminopropionic acid (Dpr), 4-aminobutyric acid and so forth;-aminoadamantane acid (Aib);-aminohexanoic acid (Aha);-aminosalicilova acid (Ava); N-methylglycine or sarcosine (the (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);-2-titillans (Thi) ; methionine sulfoxide (MSO); homoarginine (hArg); N-acetylized (AcLys); 2,4-diaminopentane acid (Dbu); 2,3-di-aminobutyric acid (Dab); p-aminophenylalanine (Phe(pNH2)); N-methylvaline (MeVal); homocysteine (hCys); homophenylalanine (hPhe); homoserine (hSer); hydroxyproline (Hyp); gemopolis (hPro); N-methylated amino acids and peptide (N-substituted glycine).
Although in most cases, the amino acids of the peptide are replaced L-enantiomeric amino acids, however, these replacements are not limited to L-enantiomeric amino acids. Thus, the definition of "mutated" or "modified" forms applies to the case when 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.
It should be noted that the present invention also addresses peptide analogues, where one or more amide linkages optionally replaced by a linkage, non-amide, prepona, called amino acids, and the preferred embodiments of the invention illustrated using peptides, however, in this regard it should be noted that in those embodiments where the described non-amide linkages used herein, the term "amino acid" or "residue" means other bifunctional components, carrier group, which in its structure is similar to the side chains of amino acids. In addition, these amino acid residues can be locked or unlocked.
In addition, one or more amide bonds can be replaced by peptidomimetics or amide-mimicking compounds that generally do not affect the structure or activity of the peptides. Suitable amide-simulating components are described, for example, Olson et al., 1993, J. Med. Chem. 36:3049.
To improve the pharmacokinetic properties of the crustal polypeptide sequence of the peptide-amplifier can be used as either N-terminal or C-terminal or N-terminal and C-terminal additions. Although the sequence of the peptide amplifier is preferably used in the form of a pair of sequences, that is, it is preferable that the hybrid polypeptides contain a sequence of the peptide-amp as I am is related peptide is present or amino, either carboxylic hybrid polypeptide. In addition, these peptides-amplifiers associated with crustal polypeptide, can be used either in direct or reverse orientation, or in any suitable combination. It should be noted that all peptides-amplifiers can be entered either at the N-Terminus or at the C-end of the crustal polypeptide. Moreover, N-, S - or N - and C-terminal position of the hybrid polypeptides can be entered multiple peptide sequences amplifier. Multiple sequence peptide-amplifier can be connected directly to each other through the same types of links that are used to join sequences of peptide-amplifier to korovou the polypeptide (see below). In addition, between one or more multiple sequences of peptide-amplifier may also be present intermediate amino acid sequence of the same type as described below. These multiple sequence peptide-amplifier typically contain from 2 to about 10 separate sequences of peptide-amp (same or different amino acid sequences), and preferably from about 2 to about 4 sequences.
Kamenoi communication although for joining peptide sequences amplifier to crustal polypeptides can be used neamine communication. Such connections are well known in the art and are, for example, any of the carbon-carbon, ester and chemical bonds, which binds the peptide sequence of the amplifier of the present invention with crustal peptide.
Typically, the sequence of the peptide-amplifier directly related to crustal polypeptide. The sequence of peptide-amplifier can also be attached to the intermediate amino acid sequence that is present between the sequence of the peptide-amplifier and crustal polypeptide. Usually the size of the intermediate amino acid sequence is from about 1 to about 50 amino acid residues, and preferably from about 1 to about 10 residues. Some types of relationships described for the binding of peptide-amplifier with crustal polypeptide, can be used to bind peptide-amplifier with an intermediate peptide.
As discussed above for peptide sequences amplifier, crustal and intermediate amino acid sequence does not have to be limited to genetically coding is/or carboxylic the resulting hybrid polypeptide can contain an amino group (-NH2- or carboxylate (-COOH), respectively. Alternative aminocore hybrid polypeptide may, for example, represent a hydrophobic group, including but limited to, carbencillin, danilou, tert-butoxycarbonyl, technology, naftolin or other carbohydrate group; an acetyl group; 9-fluorenylmethoxycarbonyl (FMOC) group; or a modified non-natural amino acid residue. Alternative carboxylic hybrid polypeptide may, for example, to represent aminogroup; tert-butoxycarbonyl group; or a modified non-natural amino acid residue. As a non-limiting example is the amino - and/or carboxylic the resulting hybrid polypeptide, which may contain any amino - and/or carboxykinase modification, illustrated in the peptides shown in Fig.13 or in the following Table 2.
Generally, the hybrid polypeptide contains the amino acid sequence, which is an unnatural amino acid sequence. That is usually the amino acid sequence of the hybrid polypeptide does not consist only of the amino acid sequence of a fragment of the endogenous nature is polypeptide.
The crust polypeptides can contain any polypeptide that can be introduced into a living system, for example, any polypeptide that can act as a pharmacologically effective polypeptide. Such cow polypeptides can be, for example, used to treat or prevent diseases or diagnostic or prognostic methods, including imaging techniques in vivo. The lower limit of the size of the crustal polypeptide is normally around 4-6 amino acid residues. Theoretically, there is no upper limit to the size of the crustal polypeptide, and this core polypeptide can contain any natural polypeptide or its fragment or any modified or synthetic polypeptide. In General, however, the size of the crustal polypeptide is from about 4-6 amino acids to about 494-500 amino acids, preferably from about 4 to about 94-100 amino acid residues, and most preferably from about 4 to about amino acid residues 34-40.
Examples of possible crustal polypeptides, which are given only for illustrative purposes, but not to limit, include, but are not limited to, growth factors, cytokines, therapeutic polypeptides, hormones such as insulin and parenterally, interleukins, chemokines, interferons, colony stimulating factors, angiogenic factors, receptor ligands, agonists, antagonists or inverse agonists, agents for the delivery of peptides, such as imaging agents, or agents for delivery of cytotoxic molecules, and extracellular matrix proteins such as collagen, laminin, fibronectin and integrin etc. in Addition, as crustal polypeptides can be used viral or bacterial polypeptides that are directly or indirectly function as immunogen or antigens, and therefore they can be used for treating or preventing pathological conditions.
Representative examples of hybrid polypeptides containing core polypeptides derived from the sequence of a viral protein, is shown in Fig.13, where these crustal sequence of the polypeptide is shaded. Core polypeptides also include, but are not limited to, polypeptides, described in U.S. Patent No. 5464933, in U.S. Patent No. 5656480 and in WO 96/19495, each of which in its entirety is introduced into the present description by reference.
The crustal sequences of the polypeptide may also be, without limitation, polethylene in Table 2, in addition to crustal polypeptides are hybrid polypeptides. However, given the sequences of the peptide-amplifier it is obvious that the crustal sequence of the polypeptide is present as part of a hybrid polypeptide.
It should be noted that the peptides listed in Table 2, also included in the scope of the present invention. As discussed above, the peptides shown in Table 2, which does not yet contain peptide sequences amplifier (that is not a hybrid polypeptides), may be used in combination with the sequences of the peptide-amplifier and are considered in this description to generate the hybrid polypeptides. In addition, the crust core polypeptides and polypeptide hybrid polypeptides shown in Table 2 and Fig.13, can be used in combination with any sequences of peptide-amplifier described in this application for routine production of other hybrid polypeptides, which are also included in the scope of the present invention.
It should be noted that, although a number of polypeptides listed in Table 2 and Fig.13, is provided with modified, for example with a blocked amino - and/or carboxykinase or D-isomer of amino aminokislotnoi sequence, presented in Table 2 and Fig.13, is also considered part of the present invention.
The crustal sequence of the polypeptide per se, shown in Table 2 and Fig.13, and hybrid polypeptides containing such core polypeptides may possess antiviral and/or antipathogenic activity and/or may have the ability to modulate intracellular processes associated with the peptide piperlime structures. Among crustal sequences of polypeptides include, for example, sequences derived from the specific sequences of the viral protein. Among crustal sequences of polypeptides include, for example, such amino acid sequences derived from more than one viral protein (e.g., a core polypeptide, derived from HIV-1, HIV-2 and SIV).
In addition, these core polypeptides can have substitutions, deletions and/or insertions of amino acids discussed above for peptide sequences amplifier, provided that they will not have a negative impact on anti-virus and/or antipathogen activity specific crustal polypeptide (as such or as part of a hybrid polypeptide).
As for deletions Amin 4-6 amino acid residues. As for insertions of amino acids, it is preferable that they amounted to no more than about 50 amino acid residues, and more preferably not more than about 15 amino acid residues. Also preferably, the insertion of core polypeptides have amino and/or carboxykinase insertions.
Among these amino - and/or carboxykinase insertion can be inserted, which include the amino acid sequence located at the amino - and/or carboxybenzoyl relative to the endogenous sequence of the protein, from which this is a core polypeptide. For example, if a core polypeptide derived from the protein gp41, this insertion must be amino - and/or carboxykinase the insertion contained in the amino acid sequence of gp41, adjacent to the crustal sequence of the gp41 polypeptide. Such amino - and/or carboxykinase insertions can mainly be about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acid residues from the amino and/or carboxylic in relation to the original korovou the polypeptide.
Hybrid polypeptides of the present invention may also contain additional modifications that make it easy to detect the polypeptide. T is well known to specialists, such methods include, but are not limited to, methods of radioactive, fluorescent or colorimetric labeling. Methods of indirect labeling is also well known in the art, and such methods include, but are not limited to, the labelling methods using Biotin/streptavidin, and indirect labeling of the antibody.
The present invention also relates to the Association of peptide sequences amplifier with molecules of other types that are not peptides. For example, the sequence of the peptide-amplifier can be attached to molecules of nucleic acids (e.g. DNA or RNA) or a small organic molecule of any type for improving the pharmacokinetic properties of these molecules.
5.2 Synthesis of peptides
Polypeptide-amplifier, a core polypeptide and the hybrid polypeptide can be synthesized or obtained by methods well known in the art. See, e.g., Creighton, 1983, Proteins: Structures and Molecular Principles, W. H. Freeman and Co., NY, which in its entirety is introduced into the present description by reference. Hybrid polypeptides can be obtained using the standard method sequential synthesis in solution or solid-phase synthesis, the condensation of f is, the work cited in this work; Solid Phase Peptide Synthesis: A Practical Approach, Atherton & Sheppard, Eds., 1989, IRL Press, Oxford, England, and works cited in this work). Similarly, can be used amino - and/or carboxykinase modification.
Polypeptide-amplifier, a core polypeptide and the hybrid polypeptide can be purified by the known methods such as normal and reverse-phase liquid chromatography, high-resolution ion-exchange chromatography, gel electrophoresis, affinity chromatography, size-exclusion chromatography, precipitation, etc., the Specific conditions used for the purification of a specific polypeptide, depend in part on the strategy of synthesis and on factors such as total charge, hydrophobicity, hydrophilicity, solubility, stability, etc., and can be selected by the specialist.
Hybrid polypeptides, the polypeptides amplifiers and core polypeptides can also be produced using techniques of recombinant DNA. In this case, the nucleotide sequence encoding the polypeptides of the present invention, can be synthesized and/or cloned and expressed by methods well known to every expert. See, for example, Sambrook et al., (1989), Molecular Cloning: A Laborat the mi molecular biology, well-known specialists. So, for example, to generate a DNA fragment encoding the desired protein may be used in polymerase chain reaction (PCR). This alternative DNA fragment can be obtained from commercial sources.
DNA encoding a desired polypeptide may be constructed by methods of recombinant DNA in a number of vector systems hosts, who also provide large-scale replication of this DNA. These vectors can be designed so that they contain the elements required for the regulation of transcription and/or translation of the DNA sequence that encodes a hybrid polypeptide.
Vectors that can be used for this purpose include, but are not limited to, vectors derived from recombinant bacteriophage DNA, plasmid DNA or kosmidou DNA. So, for example, can be used plasmid vectors such as pcDNA3, pBR322, pUC 19/18, pUC 118, 119 and vectors M13mp series. Bacteriophagous vectors can begt10,gt11,gt18-23,ZAP/R and bacteriophobia vectors series EMBL. Kominami vectors, which can be used are, but are not limited to strayaway recombinant vectors based on viruses, including, but not limited to, vectors derived from virus such as the herpes virus, retrovirus, virus, cowpox virus, adenoviruses, adeno-associated viruses or viruses, bovine papilloma or of plant viruses such as tobacco mosaic virus and baculovirus.
For the expression of biologically active polypeptides in appropriate expressing vector, i.e. a vector which contains the necessary elements for the transcription and translation of the built-in coding sequences can be incorporated nucleotide sequence encoding the protein. Designing expressing vectors having a sequence encoding a hybrid polypeptide and functionally attached to the transcriptional/translational regulatory signals may be carried out by methods well known in the art. By such methods is the technique of recombinant DNA in vitro and methods of synthesis. See, for example, the methods described in Sambrook et al., (1989), Molecular Cloning, A Laboratory manual. Cold Spring Harbor Laboratory, and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N. Y., which in its entirety are introduced in the present description by reference.
The nucleic acid molecule encoding a well is different promoter/enhancer elements. For optimization of expression of therapeutic amounts of protein can be selected corresponding to the promoter/enhancer elements. The expression elements of these vectors can vary along its length and specificity. Depending on the system host/vector may be used any of the bets transcription and translation. This promoter may be present in the form of the promoter that is naturally associated with the desired gene. This alternative DNA may be under the control of a recombinant or heterologous promoter, i.e. a promoter that is not normally associated with this gene. So, for example, tissue-specific promoter/enhancer elements can be used for regulating the expression of embedded DNA in the cells of a particular type.
Examples of transcriptional regulatory regions with tissue specificity, which have been described and which may be used include, but are not limited to, the regulatory region of the gene elastase I, which is active in the acinar cells of the pancreas (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); regulatory region of the gene of insulin, kotronopoulou, which is 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. 7:1436-1444): regulatory region of the gene of albumin, which is active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276), the regulatory region of the gene alpha-fetoprotein, which is active in liver (Krumlauf et al., 1985, Mol.Cell.Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58); regulatory region of the gene alpha-1-antitrypsin, which is active in liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); regulatory region of the beta-globin gene, which is active in myeloid cells (Magram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94); the regulatory region of the gene of the basic protein of myelin, which is active in oligodendrocyte brain (Readhead et al., 1987, Cell 48:703-712); regulatory region of the gene light chain 2 myosin, which is active in skeletal muscle (Shani, 1985, Nature 314:283-286); and the regulatory region of the gene gonadotropin hormone, which 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 7.5 K, SV40, HSV, adenovirus MLP, MMTV, LTR and CMV), as well as promoters produced with paconstitution or inducible, and can be used under appropriate conditions for the implementation of a high level of expression or carry on regulated expression of the desired nucleotide sequence. Expression of genes under the control of constitutive promoters does not require for its induction in the presence of a specific substrate, and this expression will occur under all conditions of cell growth. In contrast, the expression of genes regulated inducible promoters depends on the presence or absence of the inducing agent.
For sufficient translation built-in sequences encoding the protein, is also required specific initiation signals. Such signals are the initiating ATG codon and adjacent sequences. In cases where the relevant expressing vectors built all the coding sequence, including the initiating codon and adjacent sequences, additional signals regulation of translation is not required. However, when built only a portion of the coding sequence, requires the presence of exogenous signals, regulation of translation, including the initiating codon ATG. In addition, to ensure translation of the entire insert the initiating codon must be in the same reading frame as the sequence encoding the protein. These exogenous signals regulation of translation initiator codons can is wycena by including sequences of transcription attenuators, enhancer elements, etc.,
5.3. The sequences of the peptides of usilitelei, crustal polypeptides and hybrid polypeptides of the present invention
As discussed above, the sequence of the peptide-amplifier can be used to improve the pharmacokinetic properties of any crustal polypeptide by incorporating a specified crustal polypeptide to the sequence of the peptide-amplifier with the formation of the hybrid polypeptide. Such an improvement of pharmacokinetic properties is observed in comparison with the pharmacokinetic properties of the crustal polypeptide, taken separately. Standard parameters pharmacokinetic properties and methods for the determination and characterization of the pharmacokinetic properties of the agent, as the polypeptide, are well known in the art. Limitiruyuschie examples of such methods are presented in the following Examples.
The sequence of peptide-amplifier of the present invention, furthermore, can be used to increase the in vitro or ex vivo time-life crustal polypeptide, to which may be attached sequence peptide-amplifier. For example, the sequence of the peptide amplifier can increase the time progesteronee culture, tissue culture or in samples taken from the patient (for example, in the sample of cells, tissue samples for biopsy or other samples containing body fluids).
Core polypeptides and hybrid polypeptides of the present invention can also be used in methods of modulating (e.g., reducing, inhibiting, resolve, stabilize, or enhance) fusogenic events. These peptides preferably have antipathogenic or antiviral activity. The peptides of the present invention may also have the ability to modulate intracellular processes, including interactions associated with peptide bierley structure.
In a specific embodiment of the invention, the hybrid polypeptides and core polypeptides of the present invention, which possess antiviral activity, can be used in an activated methods. Such activated methods can be used, e.g., against human retroviruses, such as HIV (human immunodeficiency virus), such as HIV-1 and HIV-2, and against T-lymphotropic human viruses (HTLV-I and HTLV-II), and non-human retroviruses, such as leukemia virus cows, viruses, sarcoma, and leukemia cats, viruses immunobased of the invention can also be used against viruses, non-retroviruses, including, but not limited to, respiratory syncytial virus (RSV), a virus canine plague, the virus newcastlegay disease, parainfluenza virus human influenza viruses, measles viruses, viruses, Epstein-Barr, hepatitis b viruses and viruses Mason-Pfizer.
The above viruses are viruses with the shell. Anti-virus methods of the present invention can also be used against viruses without shell, including, but not limited to, picornaviruses, such as poliovirus, hepatitis a virus, enterovirus, Echovirus and Coxsackie virus, papovavirus, such as the human papilloma virus, parvoviruses, adenoviruses and reoviruses.
Other types antipathogenic activity that can be modulated by the methods of using the peptides of the present invention, include, but are not limited to, modulating the metabolism of neurotransmitters by merging cells and fusion of sperm with the egg. From the intracellular violations, including interaction due to bierley structure of peptides, which can be weakened ways using the peptides of the present invention are, for example, disorders caused by bacterial toxins.
Antifungalen routine methods using standard in vitro and ex vivo assays and assays using animal models of this disease, which, from the point of view of antiviral activity, can be specific or partially specific to the desired virus and which are well known to specialists.
Vicepresidencia description mainly refers to the anti-virus and antipathogenic activity crustal and hybrid polypeptides of the present invention. Hybrid polypeptides of the present invention can also be used in any method which provides for the introduction or use of the crustal polypeptide, taken separately. The use of hybrid polypeptides in such methods is particularly preferred in cases where it is necessary to improve the pharmacokinetic properties of the crustal polypeptide. For example, in the treatment of some types of diabetes used insulin. Therefore, a hybrid polypeptide containing insulin or a fragment of insulin in the form of crustal polypeptide, can also be used in the methods of easing the symptoms of some forms of diabetes, using and/or provides for the use of insulin.
In addition to the above therapeutic methods, the peptides of the present invention can be also used in the prognostic methods for con is nutriclean processes, associated with piperlime peptide structures; viral infection caused by cell fusion and/or fusion of the virus with the cell. For example, the crust and hybrid polypeptides of the present invention can be used in prophylactic methods to prevent viral infections.
Hybrid polypeptides of the present invention, furthermore, can be used in diagnostic methods. Such methods can be in vivo or in vitro methods. Any diagnostic method, which can be used in specific core polypeptide, can be carried out using a hybrid polypeptide containing this core polypeptide and the modification or the primary amino acid sequence, which allows the detection of the hybrid polypeptide. Such methods, in comparison with the diagnostic methods may have advantages, namely, that increased the half-life of the hybrid polypeptide in comparison with the crustal polypeptide, taken separately, can enhance the sensitivity of the diagnostic method in which it is used. Such diagnostic methods include, but are not limited to, imaging techniques, such as mitotic hybrid polypeptide, can be detected by binding hybrid polypeptide and visualization (direct or indirect method) this associated hybrid polypeptide.
5.4. Pharmaceutical compositions, doses and routes of administration
The peptides of the present invention can be introduced by methods well known in the art. Appropriate agents are preferably produced and administered systemically. Description of how the finished compositions and their administration can be found in the manual "Remington''s Pharmaceutical Sciences, latest edition. Mack Publishing Co., Easton, PA. Suitable routes of administration may be oral administration, rectal administration, vaginal introduction, introduction intra-lungs (by inhalation), transcutaneous introduction, introduction through the mucous and putting in the introduction; parenteral administration, including intramuscular, subcutaneous, intramedullary injections, as well as vnutriobolochechnoe, direct intraventricular, intravenous, intraperitoneal, intranasal or ophthalmic injection, etc. For intravenous injection, the agents of the present invention can be manufactured in the form of aqueous solutions, preferably in a buffered physiological solution such as Hanks solution, ringer's solution Z to be used in infusion pumps. For insertion through the mucous membrane in this composition are used penetrants, facilitating the passage of the peptide through the barrier. Such penetrants, basically well known to the experts.
In cases where it is preferable intracellular introduction of the peptides of the present invention or other inhibitory agents that can be used by methods well known in the art. For example, such agents can be agents encapsulated in liposomes or microspheres, which are then injected as described above. Liposomes are spherical of lipid bilayers with an aqueous core. This water entered the core of all the molecules present in aqueous solution in the process of forming liposomes. Liposomes are protected from external microenvironment, and, thanks to the fusion of liposomes with the cell membrane, it can be effectively delivered into the cytoplasm of the cell. In addition, if you want to enter small molecules, due to the hydrophobicity of these liposomes can be made direct intracellular introduction.
The nucleotide sequences encoding the peptides of the present invention, intended for intracellular introduction, can be expressed by the s-based viruses such as retroviruses, viruses cow pox, adeno-associated viruses, herpes viruses, or viruses bovine papillomavirus, can be used to deliver and expresii such nucleotide sequences in the desired cell population. Methods of constructing such vectors and expression constructs are well known in the art. 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 and Wiley Interscience, NY.
The effective dose of the peptides of the present invention can be determined by methods well known in the art, in accordance with such parameters as biological half-life, bioavailability and toxicity. In particularly preferred versions of the invention, the effective dose of the hybrid polypeptide is at intervals that can be defined by the expert, on the basis of data obtained from routine in vitro and in vivo studies, is well known to specialists. For example, analyses of the cells in vitro-cultures with anti-virus activity, such as the assays described below in Section 7 for T, provide data from which each spetsializirovany certain amount of infectivity of the virus (for example, 50%, IC50; or 90% IC90). Then, using pharmacokinetic data, such as pharmacokinetic data described below in Section 10 for T and obtained from one or several experimental animal models of this disease, the specialist can be selected corresponding to the dose so as to obtain the minimum concentration of peptide in plasma (Cmin), which is equal to a certain value IC or exceeds this value.
As example can serve as a dose of polypeptides that comprise at least 0.1 μg/kg of body weight and up to 10 mg/kg of body weight. Preferred effective dose is from 0.1 to 100 μg/kg of body weight. As other examples can serve as a dose of the peptides of the present invention, the components of 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. The term "therapeutically effective dose" means an amount of compound sufficient to achieve attenuation of symptoms or to extend the life of the patient. Toxicity and therapeutic efficacy of such compounds can be determined using standard therapeutic procedures carried out on cell cultures or experimental animals, NAPA for 50% of the population). The ratio of doses for toxic and therapeutic effects is a therapeutic index that can be expressed as the ratio LD50/ED50. While preferred are compounds which have a high therapeutic index. To establish intervals of doses for administration to humans can be used data obtained from analyses of cell cultures and animal study. Doses of such compounds preferably are such quantities of these compounds to their concentration in blood was ED50with insignificant toxicity or lack of it. These doses may vary within these limits depending on the form of the medicinal product and method of its introduction. For any compound used in the method of the present invention, therapeutically effective dose can be initially evaluated by analysis of cell cultures. A certain dose can be injected animal model of this disease to achieve a plasma concentration that is inhibitory concentration IC50(for example, the concentration of the test compound, which leads to polymyxine the structure to the number of events merge in the absence of the test compound), defined in cell culture. Such data can be used for more accurate determination of effective doses for administration to man. Concentration in plasma may be measured, for example, using high-performance liquid chromatography (URGH) or any biological or immunological analysis, which can be defined levels of the peptide.
Hybrid polypeptides of the present invention can be introduced in the form of one-time or fractional doses, periodically or continuously. For example, the polypeptides of the present invention can be entered as a single dose, such as a single dose, injected subcutaneously, single dose, administered by intravenous infusion, or a single dose, administered orally. The polypeptides of the present invention can also be entered multiple times in multiple doses, including periodic introduction. For example, in some embodiments of the invention, the polypeptides of the present invention can be injected once a week, once a day, twice a day (for example, every 12 hours, every 6 hours, every 4 hours, every 2 hours or 1 hour. The polypeptides of the present invention can be implemented in continuous R is subcutaneous or other implant, which leads to a continuous absorption of these polypeptides in the patient's body.
Hybrid polypeptides of the present invention can also be introduced in combination, at least one other therapeutic agent. This can be used with other types of therapy, although they are not preferred for HIV therapy (e.g. cancer therapy), and these therapies can be carried out simultaneously or sequentially, including cyclic therapy (i.e., the introduction of the first connection during a certain period of time with subsequent introduction of a second antiviral compound within a certain period of time, and then repeating this sequential injection to minimize the possibility of development of resistance to one of these therapies).
In case of a virus, such as retroviral, infection can be introduced effective amount of the hybrid polypeptide or its pharmaceutically acceptable derivative in combination with at least one, and preferably, at least two other anti-viral agents.
If you take HIV infection, such anti-viral agents can be, but are not limited IMI another hybrid polypeptide, a core polypeptide which is at least partially derived from HIV-1 or HIV-2, cytokines, for example, pIFN, pIFN, pIFN; reverse transcriptase inhibitors, including nucleoside and non-nucleoside inhibitors, such as AZT, 3TC, D4T, ddI, adefovir, abacavir or other dideoxynucleoside or dimethoxyphenylacetic, or mesilate of delavirdine, nevirapine, afaires; inhibitors of capping viral mRNAs, such as ribavirin; HIV protease inhibitors such as ritonavir, nelfinavir mesilate, APV, SQV, saquinavir mesilate, indinavir or AWT, AWT or MC; amphotericin b as Lipetskaya molecules with activity against HIV; and from castanospermine as an inhibitor of glycoprotein processing.
Hybrid and/or core polypeptides of the present invention, furthermore, can be used for preventive purposes to prevent diseases. Hybrid and/or core polypeptides may act directly to prevent diseases, or, alternatively, they can be used as vaccines, which stimulate production of host antibodies against hybrid polypeptid, including, for example, inhibition of viral, bacterial or parasitic infection.
For all kinds of treatment described above, the specific composition, the route of administration and dosage can be chosen by the attending physician based on the patient's condition (see, e.g., Fingl et al., 1975, The Pharmacological Basis of Therapeutics, Ch.1, p.1).
It should be noted that the attending physician must know how and when should it be completed, terminated or adjusted the administration of a medicinal product because of its toxicity or organ dysfunction. Conversely, the attending physician must know how and when treatment should be adjusted in the direction of increasing the injected doses, if not achieved adequate clinical response (with the exception of toxicity). The magnitude of the injected dose in the treatment of this oncogenic disorder may vary depending on the severity of the disease of the patient being treated, and the route of administration. Dose and, probably, the introduction may also vary depending on the age, body weight and sensitivity of each individual patient. The program, similar to those described above, can be used in veterinary medicine.
In the scope of the present invention includes the use of pharmaceutically sistemnogo introduction. With proper choice of carrier and suitable manufacturing method, the composition of the present invention, particularly compositions obtained in the form of solutions, can be injected parenterally, for example, by subcutaneous injection, intravenous injection, subcutaneous infusion or intravenous infusion, for example, by a pump. These compounds can be easily manufactured using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers suitable for administration of the compounds of this invention are tablets, coated tablets, capsules, liquids, gels, syrups, slurries, suspensions, etc., used for oral administration by the patient.
Pharmaceutical compositions suitable for use in the present invention are compositions where these active ingredients are contained in amounts effective to achieve the desired goal. Each specialist can determine the effective amount of the active ingredient, particularly on the basis of the detailed descriptions given in this application.
In addition to the active ingredients, these pharmaceutical compositions may contain suitable headlight is tion of these active compounds, drugs, which can be used for pharmaceutical purposes. Preparations for oral administration can be produced in the form of tablets, pills, capsules or solutions. For oral administration of peptides can be used in the methods described, for example, Emisphere Technologies, which are well known and are typically used by professionals.
The pharmaceutical compositions of the present invention can be manufactured by methods known in essence, for example, by means of simple mixing, dissolving, granulating, production drops, grinding into powder, spray drying, emulsification, encapsulation, seizing or lyophilization.
Pharmaceutical compositions for parenteral administration are aqueous solutions of the active compounds in water-soluble form. In addition, emulsions and suspensions of the active compounds can be obtained in the form of suitable oil blends for injection. Suitable lipophilic solvents or carriers are fatty oils such as sesame oil, or synthetic esters of fatty acids, such as etiloleat or triglycerides, liposomes or other substances used by the experts for the preparation of lipid or lipophilic the emulsion is tricoderma carboxymethylcellulose, sorbitol, or dextran. To obtain solutions with a high concentration of these suspensions may also contain, but not necessarily suitable stabilizers or agents which increase the solubility of the compounds.
Pharmaceutical preparations for oral administration can be obtained by combining the active compounds with solid excipient, optionally grinding the resulting mixture and the mixture of granules, after adding suitable additives, if necessary, to obtain core for tablets or pills. Suitable carriers are in particular 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, tragacanth gum, methylcellulose, hypromellose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone (PVP). If necessary, can be added dezintegriruetsja agents, such as polyvinylpyrrolidone cross, agar or alginic acid or a salt thereof such as sodium alginate.
The core tablets are covered with suitable coatings. For this purpose, can the gum, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and/or titanium dioxide, solutions varnishes and suitable organic solvents or solvent mixtures. To identify or characterize different combinations of doses of active compounds in the coating of tablets or pills may be added dyes or pigments.
Pharmaceutical preparations which can be used for oral administration are capsules with device to eject, made of gelatin, and also soft sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Capsules with a device for ejection may contain active ingredients mixed with a filler, such as lactose, with binding agents such as starches, and/or oiling agents, such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, can be added stabilizers.
In cases where it is necessary to increase the immune response in the host, the hybrid polypeptides can be manufactured is not limited to, mineral gels such as aluminum hydroxide; surface active substances such as lysolecithin, the polyols of the type pluronics, polyanion; other peptides; oil emulsions; and potentially suitable adjuvants such as BCG and Corynebacterium parvum. For the introduction described in this application, the vaccine compositions can be used many ways. Such methods include, but are not limited to, oral, percutaneous, intramuscular, intra-lungs, intravenous, subcutaneous and intranasal administration.
6. Example: Identification of a consensus amino acid sequence containing the sequence of the peptide-amplifier
Protein gp41 retrovirus contains structural domains called the area-helix located at the C-terminal region of the protein, and the area latinboy lightning", located in the N-terminal region of the protein. A comparative analysis of the plots of primary sequences of the peptide-amplifier, derived from gp41 (Fig.2A and 2B) with gp41 for all currently published identified a consensus amino acid sequences of isolates of HIV-1, HIV-2 and SIV, illustrated in Fig.1.
As described in detail in negativeone Examples such settlement is innych sequences to other diverse crustal polypeptides leads to improved pharmacokinetic properties of the resulting hybrid polypeptide.
7. Example: Hybrid polypeptides that function as strong inhibitors of HIV-1 infection
T shown in Fig.13 is a hybrid polypeptide containing the sequence of the peptide amplifier attached to korovou the HIV polypeptide. As demonstrated below, the hybrid polypeptide T has improved pharmacokinetic properties and strong activity in vitro against isolates of HIV-1, HIV-2 and SIV, with increased activity against clinical isolates of HIV-1 was observed in in vitro assays on the degree of infection with HHRUMS, as well as in in vivo studies on HIV-1 infection in a murine model of SCID-HuPBMC. In the biological assays described above, the activity T was comparable to the strong antiviral activity of the polypeptide T20. The T20 polypeptide, also known as DP-178 and obtained from the sequence of a protein gp41 of HIV-1 has been described and claimed in U.S. patent No. 5464933.
7.1. Materials and methods
7.1.1. Synthesis and purification of peptides
Peptides were synthesized using the rapid-MOC chemistry. Basically, unless specifically indicated, these peptides contained amidarone carboxylic and the acetylated aminocore. Purification was performed using reverse-phase HPLC.
T (Ac-WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF-NH22). Thus, T is a core polypeptide hybrid polypeptide T. T blocked at its carboxy - and aminobenzo as T.
In particular, T synthesized by standard methods of solid-phase synthesis. The identity of the main peak on the HPLC-chromatogram, which was confirmed by mass spectroscopy, indicated T.
T was easily purified using reverse-phase chromatography on a 6-inch column Packed with C18, 10 micron, the substrate 120.
Virus HIV-1LAI(M. Popovic et al., 1984, Science 224:497-508) were propagated in CEM cells, cultured in RPMI-1640 containing 10% fetal calf serum. Supernatant from infected CEM cells was passed through the 0.2 micron filter and the titer of infectivity was assessed by analysis on microinjections using cell lines AA to support virus replication. For this purpose, 20 μl of serially diluted virus was added to 20 μl of CEM cells at a concentration of 6 x 105/ml in 96-Aravali within 7 days by adding fresh medium every other day. 7 days after infection samples supernatants tested for viral replication, as evidenced by the reverse transcriptase activity released in the supernatant. TCID50was calculated by the formula of reed and Manisa (Reed L. J. et al., 1938, Am. J. Hyg. 27:493-497).
7.1.3. Analysis of cell fusion
Approximately 7104cells Molt-4 were incubated with 1 x 104the CEM cells chronically infected with HIV-1LAIin 96-well tablets for the cultivation of tissues in a final volume of 100 μl culture medium (RPMI-1640 containing 10% thermoinactivation FBS plus 1% L-glutamine and 1% Pen-Strep), described previously (Matthews, T. J. et al., 1987, Proc. Natl. Acad. Sci., USA, 84:5424-5428). Peptide inhibitors were added in a volume of 10 μl, and the cell mixtures were incubated for 24 h at 37With 5% CO2. At this stage polynuclear giant cells (syncytium, width 5 cells or more were counted under the microscope at magnification of 10x and 40x, which allowed to visualize the entire hole in one field. The treated cells were compared with infected untreated control cells, and the results were expressed as percentage of inhibition from the infected control.
7.1.4. Analyses on infectionmost the social Institute of health (NIH) research SPID (NIH AIDS) and standard reagents, Department SPID and the national Institute of arthritis and infectious diseases (NIAID; C. Chackerian et al., 1997, J. Virol. 71:3932-3939) were seeded into 48-well plate for culturing tissue (approximately 2104cells/well in a volume of 300 µl/well of a selective culture medium consisting of DMEM with addition of 10% terminateserver FBS, 1% L-glutamine, 1% Pen-Strep, hygromycin, geneticin and puromycin) and left overnight for binding at 37With 5% CO2. The next day, confluently cells was approximately 30%. Environment for sowing was removed and added to the dissolved peptide inhibitor in volumes of 50 µl/well (untreated control - only environment), and then added 100 μl/well of diluted virus (desired input titer of virus = 100-200 b.about.E./well). Finally, each well was added 250 μl of selective culture medium, and the plate is incubated for 2 days at 37With 5% CO2. Fixation and staining were performed according to the Protocol provided by the National Institute of arthritis and infectious diseases, and using cells MAGI-CCR5. For this, the medium was removed from the tablet, and to each well was added 500 μl of fixat the plants were washed in DPBS and to each well was added 200 μl of staining solution. The tablet then incubated at 37With 5% CO2within 50 minutes, the dye solution was removed and each well was twice washed in DPBS. The tablet was left for air drying, and then blue cells were counted on a microscope, all wells were numbered. Treated wells was compared with the infected untreated control cells, and the results were expressed as percentage of inhibition from the infected control.
7.1.5. Analysis by reverse transcriptase
Microanalysis for reverse transcriptase (RT) was performed in an adapted method Goff et al. (Goff, S. et al., 1981, J. Virol. 38:239-248) and Willey et al. (Willey, R. et al., 1988, J. Virol. 62:139-147). Supernatant from virus/cell cultures brought up to a volume of 1% Triton-X100. 10 ál of each sample supernatant/Triton-X-100 was added to 50 μl of RT mixture (75 mm Cl, 2 mm reagent Clevelands, 5 mm MgCl2, 5 μg/ml poly A, and 0.25 units/ml oligo dT, 0,05% N40, 50 mm Tris-HCl, pH of 7.8, 0.5 µm of non-radioactive dTTP and 10 ski/ml32P-dTTP) in 96-well microtitration tablet with a U-shaped bottom, and incubated at 37C for 90 minutes. After incubation with 40 μl of reaction mixture from each well was transferred into the apparatus for dot-blotting Schleicher & Schuell (S+S) in a partial vacuum containing sets M sodium citrate). Each well was washed 4 times, at least 200 ál of 2x SSC in a complete vacuum. Filter (Minifold) was dismantled and mesh filter paper was removed and 3 times were washed in 2x SSC. Finally, the filter membrane was trenirovki on absorption paper left on the drying air and placed in bags, which was tightly sealed. The samples were placed in a cassette with a phosphor screen, "washed" applied luminescent screen (at least 8 minutes) and was closed. Akinyosoye was 16 hours. Index values of pixels (PIV) generated in the presentation of data on the volume, selected on the basis of fluorescent imaging (Molecular Dynamic Phosphorimager) blots were used to identify damaged or inhibited fraction (Fa) for all doses of the inhibitor(s) by comparing with the untreated infected control (analyzed using data on the volume ImageQuant adjusted for background values).
7.1.6. The analysis on an infection/neutralizing human MCPC (HhRVMS)
We used the prototype analysis, where the cell lines used primary isolate cells MCPC obtained from Interstate blood Bank and activated within 2-3 days ACTS (0.5 μg/ml) in SM), washed and froze. The cells were thawed, if necessary, and prior to analysis activated as described above at least 2-3 days. In this assay using 96-well pad cell concentration was 2106/ml in the medium of 5% IL-2, and the final volume was 100 μl. The original peptide solutions were prepared in DPBS (1 mg/ml). Cultivation of peptides was carried out in 20% FBS-RPMI-1640/complete medium with 5% IL-2.
7.1.7. HU-PBMC-SCID model of HIV-1 infection in vivo
The SCID female mice (aged 5-7 weeks) was intraperitoneally injected with 5-10 x 107MCPC adult. Two weeks after recovery, mice on day 0 infitsirovali intraperitoneally (i.p.) HIV-1 9320 with 103TCID50(isolate A susceptible to AZT). The processing of the peptides was carried out i.p. twice a day, starting from day -1 and continuing for 6 days. The degree of infection of blood cells, splenocytes, lymph node and peritoneal cells were evaluated every week for the next three weeks with quantitative cultivation blasts human MCPC, then the animals were killed by exsanguination, and tissues were collected (on day 7, approximately 12-18 hours after the last treatment drug). Supanburi and Protocol Immunotek Coulter).
7.1.8. Pharmacokinetic studies in rats
Were used 250-300 g male rats of the CD, which was introduced double neck catheter, obtained from Charles River Laboratories. In one of the cervical catheter were injected with the peptide in a volume of 200 μl of peptide solution (approximately 3.75 mg/ml), the dose concentration of the solution was determined by the method of Adelkhah (Edeihoch, 1967, Biochemistry 6:1948-1954) and corrected based on the weight of the animal so that each animal received a dose of 2.5 mg/kg at predefined intervals of time (0, 15, 30 min and 1, 2, 4, 6 and 8 hours) took approximately 250-300 μl of blood was added to Capiject tubes with EDTA. From the besieged cells after centrifugation was removed plasma, and this plasma was either frozen or used immediately for fluorescence HPLC analysis.
7.1.9. Fluorescent URGH-analysis of plasma samples
100 μl plasma samples were added to 900 μl of buffer for precipitation (acetonitrile, and 1.0% TFA, detergent), which led to the deposition of most of the plasma proteins. After centrifugation at 10,000 rpm for 10 minutes, 400 μl of supernatant was removed and added to 600 μl of water, suitable for HPLC. Serial dilution was performed in a buffer for cultivation, consisting of 40% buffer for precipitation and 60% water, th is the conduct of the sample, serial cultivation or dosing solution was carried out in buffer and in plasma and used to plot a standard curve of peak area for known concentrations of the peptide. This curve is then used to calculate the concentration of peptide in the plasma taking into account all the dilutions and the number of peptide introduced into the column.
7.1.10. The Protocol HTT
To measure the cytotoxic/cytostatic action of the peptides was carried out by analysis of HTT (Weislow, O. S. et al., 1989, J. Natl.Cancer Inst. 81: 577-586) in the presence of various concentrations of peptide for efficient determination of the selective index (SI). In this analysis TC50was determined by incubating cells in the presence and absence of serially diluted peptide followed by the addition of XTT. In survivors/metabolizing cells HTT restored to soluble HTT-formazane with brown color. The optical density was detected, and to determine TC50comparing the values obtained in the presence and absence of peptides using the method of Karber (see, for example, Lennette, E. H. et al., eds., 1969, Diagnonostic Procedures for Viral and Rickettsial Infections, American Public Health Association, Inc., fourth ed., pp.47-52). Cells Molt 4, SEM (80000 cell/well) and the combination of ClearCase in total volume of 100 μl. After incubation each well was added 25 μl of the working solution of XTT (1 mg/ml XTT, 250 μm PMS in complete medium containing 5% DMSO) and the plates were incubated at 37C. the color Development was recorded, and the results used to calculate the values obtained from wells containing peptide, in the form of a percentage of the untreated control wells.
7.2.1. Antiviral activity tests for the merger
T directly compared with T20 analyses on viruspositive merge cells conducted using chronically infected CEM cells, mixed with uninfected cells Molt-4, as shown below in Table 3. Inhibition of the polypeptide T merge cells, mediated laboratory isolates, such as IIIb, MN and RF was comparable to the inhibition of the polypeptide T20 and approximately 2.5-5 times higher than the inhibition merge cells, the polypeptide T20. T was also more active (3-28 times) than T20, in relation to several syncytiainducing clinical isolates, including AZT-resistency isolate (G691-2) pre-AZT-treated protein isolate (G762-3) and isolate 9320 (used in the research model HuPBMC-SCID). It should be emphasized that T was osti - analyses on the degree of infection Magi-CCR-5
Analyses on an infection Magi-CCR-5 allow direct comparison of syncytial and asyncfile inducing viral isolates, as well as comparisons between laboratory and clinical isolates. This analysis also allows to directly set the degree of virus infection (expression of TAT after infection, LTR transactivation, leading to the production of beta-galactosidase) in comparison with the commonly used indirect methods of measurement of infectivity, such as the analysis on the production of antigen P24 or reverse transcriptase. Analyses on the degree of infection Magi-CCR-5 (see Table 4 below) revealed that T is consistently more effective than T20 for all tested isolates, on the basis of calculation of parameters of inhibition, EU50and Vn/Vo=0,1. It was found that T is a much stronger inhibitor (>25 times) against clinical HIV-1 isolate 301714, which is one of the least sensitive isolates for T20. In addition, T, at least, is 100 times stronger inhibitor than T20, in respect of the isolate HIV W. These data together with data merge suggest SNA activity - analyses on the degree of infection MCPC man
T directly compared with T20 analyses on the degree of infection MCPC person (see Table 5, below), which are identifiable surrogate in vitro system for the pre-concentration of the drug in the plasma is required for inhibition of the virus in vivo. These comparisons showed that T is a stronger agonist against all isolates, HIV-1, tested at the present time, all values of Vn/Vo=0,1 (dose required to reduce the titre of virus in one log) were reduced to submicrogram concentrations. Many of the clinical isolates, the least sensitive to T20, was found 10 times or greater sensitivity to T. It should be emphasized that HIV-1 9320, that is, the isolate used to infect murine models HuPBMC-SCID, was 46 times less sensitive to T20 than T, indicating a very good correlation with in vivo results.
7.2.4. Antiviral activity - T20-resistant laboratory isolates (LAB isolates)
T directly compared with T20 analyses on viruspositive merge cells conducted using ruival almost 200 times more active against T20-resistant isolate, than T20.
In the analysis of Magi-CCR-5 (see below Table 7) T found in 50,000 times higher activity than T20, T20 against resistant isolates, such as pNL4-3 SM and pNL4-3 STM (Rimsky, L. & Mattews, T., J. Virol. 72:986-933).
T directly compared with T20 analyses on the degree of infection MCPC person (see below Table 8) to assess differences in their activities against drug-resistant isolates. T found in almost 250 times more active than T20, T20 against resistant isolates pNL4-3 SM.
7.2.5. Antiviral activity - HU-PBMC-SCID model in vivo
Anti-virus in vivo activity T directly compared with the activity of T20 in mouse models HuPBMC-SCID infected with HIV-1 virus 9320 (Fig.3). Two weeks after recovery, mice with MCPC man these mice intraperitoneally on day 0 (i.p.) infected isolates from HIV-1 9320 with TCID50103(isolate A susceptible to AZT), passaged on MCPC. The processing of the peptides was carried out i.p. twice a day 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 from day -1. The degree of infection of blood cells, splenocytes, lymph node and peritoneal knii with blasts of human MCPC, then the animals were killed by exsanguination, and tissues were collected (on the 7th day, after about 12-18 hours after the last treatment drug). Supernatant from this co-culture was assessed on the production of antigen P24 HIV-1 as an indicator of viral infection. Infectious virus was not detected in the tissues of the blood or lymph T20-treated animals, however, this virus was detected in the peritoneal washes and medications spleen. All compartments were negative for infectious virus at a dose of 6.7 mg/kg of the polypeptide T that pointed to its higher activity at least 10 times greater than the activity of T20. At the dose of 2.0 mg/kg T and in the lymph, and spleen was not detected infectious virus, and in the peritoneal washes were observed reduction of 2 log10the titer of virus in the blood was observed a reduction of 1 log10the titer of the virus compared to the infected control. At the lowest dose T, that is to 0.67 mg/kg, peritoneal washing, and blood were equivalent infected control; however, in the tissues and lymph in the tissues of the spleen was observed, at least, the reduction of 1 log10the titer of infectious virus. In General, these results showed that when these uski rats
For additional definitions pharmacokinetic profile T were used kanalirovanie rats. Male CD rats, 250-300 g, intravenous (i.v.) introduced T and T20 through the jugular catheter (Fig.4A-5). The resulting plasma samples were evaluated using fluorescence HPLC to estimate the number of extracted peptides in plasma. The half-life of the beta phase and total area under the curve (AUC) for T were almost three times higher than for T20 (Fig.5).
As shown in Fig.6, no cytotoxicity T in vitro were found.
In addition, T did not show strong toxicity (fatal within 24 hours) at a dose of 167 mg/kg (highest tested dose), administered i.v. through the cervical cannula (0.3 ml in 2-3 minutes).
7.2.8. Direct linking design gp41 M41178
T1249 was subjected to radioactive tagging125I, and HPLC purification to obtain the maximum specific activity. T20 was madirovalo the same way. Linking to a state of saturation with M41178 (a hybrid protein with a truncated ectodomain gp41, not containing the amino acid sequence T20) immobilized on microtiter tablets at 0.5 mg/ml, illustrated by the FIA. Specific binding was represented by the difference between total and nonspecific binding. The results showed that125I-T1249 andl25I-T20 have similar affinity binding for 1-2 nm. Graphs of inverse linear dependence of Scatchard suggest that each ligand binds to sites of homogeneous class.
The kinetics of binding125I-T1249 and125I-T20 was determined on a microtiter plate scintillation tablets coated with 0.5 μg/ml M41178. Time spent on Association and dissociation, as shown in Fig.8. The dissociation of the bound radioligand was determined after addition of unlabeled peptide to a final concentration of 10 μm in 1/10 of the total volume of analysis. The initial rate of Association and dissociation for125I-T1249 were significantly lower than for the125I-T20. The nature of dissociation for both radio was unchanged in the case when dissociation was initiated from the other unlabeled peptide (i.e.125I-T1249 with T20).
To further illustrate the fact that both ligand compete for binding to the same target site, unlabeled T and T20 was titrated in the presence of the same concentration of either125I-T1249, or125I-T20. Ligandprotein in Fig.9 and suggest although both have similar ligand affinity, however, to ensure full competition for binding with125I-T1249 required higher concentrations of either unlabeled T20 or T.
7.2.9. Direct binding HR1-region of gp41
To measure the secondary structure T in solution (phosphate buffered saline, pH 7), taken separately and in combination with a peptide of 45 residues (T) deriving from connecting areas HR1 (7-membered repeat 1) gp41, used spectroscopy circular dichroism (CD). In Fig.14A shows the CD spectrum of one T in solution (10 μm, 1C). This spectrum is typical range for peptides with alpha-helical structure. In particular, the deconvolution of this spectrum using singular value decomposition with the main spectral series for the 33 proteins allows to predict the content of a spiral in T (taken separately in solution) of 50%. In Fig.14C illustrates a characteristic CD spectrum T mixed with T. The shaded squares (n) denote theoretical CD spectrum predicted for "non-interactive" model, where the hypothesis that these peptides do not interact in solution. The actual experimental range (l) C the two peptide really interact with the formation of the measured structural changes observed in the CD spectrum.
7.2.10. Protection from protease region binding T in GP41
Susceptibility chimeric protein M41178 described above in section 7.2.8, to cleavage by proteinase-K, were determined and analyzed using polyacrylamide gel electrophoresis. The results are illustrated in Fig.15.
In a separate incubation or M41178 (raw; Fig.15, lane 2), or T (raw; Fig.15, lane 4) with proteinase K (Fig.15, lanes 3 and 5, respectively) both of them were split. However, incubation T with M41178 before adding proteinase-K (Fig.15, lane 7) resulted in obtaining protected HR-1 fragment of approximately 6500 daltons. Sequencing of the protected fragment showed that it corresponds to the region of primary sequence, localized in ectodomain gp41. This protected portion includes a soluble peptide HR1 (T) used in studies of CD, as described in section 7.2.9, and, in addition, contains an additional seven amino acid residues localized on aminocore. This protection can be an indicator of binding T with a specific sequence of gp41, which is present in construc
This example describes the hybrid polypeptides with respiratory syncytial virus (RSV), and improved pharmacokinetic properties. In addition, the results presented below, showed that the hybrid polypeptides derived from RSV, are strong inhibitors of RSV infection.
8.1. Materials and methods
8.1.1. Synthesis and purification of peptides
RSV-polypeptides were synthesized using standard chemical Express-Mos methods. Basically, if it is not specifically mentioned, these peptides contain amidarone carboxylic and the acetylated aminocore. Purification was performed using reverse-phase HPLC.
8.1.2. Analysis to reduce the number of plaques formed under the action of respiratory syncytial virus
All the necessary dilution of the peptides was carried out in a clean, sterile 96-well tablets TC. Data were collected for all eleven dilutions of each peptide and one control wells containing no peptide. The interval of the final peptide concentrations ranged from values of 50 µg/ml or 100 μg/ml to values corresponding to all eleven twofold dilutions. RSV was obtained at a concentration of 100 b.about.E./well in 100 μl of 3% of EMEM determined using known Ner was removed Wednesday. The material of the tablets for cultivation transferred to the plates with cells, beginning with row 1, and then transferred the number 12, number 11 and so on up until all the rows have not been transferred. The tablets were again placed in the incubator for 48 hours.
These cells were tested for the presence of syncytium in the control wells. Then medium was removed and to each well was added approximately 50 μl of 0.25% crystal violet in methanol. To remove excess dye wells immediately washed with water and left for drying. The number of entities in each well was counted using preprofile magnifier.
8.2. The results:
Pharmacokinetic studies of hybrid RSV-peptides T (AC-WQEWDEYDASISQVNEKINQALAYIREADELWAWF-NH2and T (Ac-WQAWDEYDASISQVNEKINQALAYIREADELWAWF-NH2), containing the sequence of the peptide amplifier, found a significantly longer half-life compared to crustal peptide T (Ac-VYPSDEYDASISQVNEEINQALAYIRKADELLENV-NH2), shown in Fig.10A-10B. Hybrid polypeptides T, T and T (Ac-WQAWDEYDASISDVNEKINQALAYIREADELWEWF-NH2) also showed a significant increase in the time half-life compared to crustal peptide T (Ac-DEYDASISQVNEKINQALAYIREADEL-NH2).
Hybrid RSV-polypeptides T, T and T, as well as polypeptides T and T were tested for their s hybrid RSV-polypeptide, and a core polypeptide T, possessed the ability to inhibit RSV infection. Unexpectedly it was found that the hybrid polypeptide T is a compound which has a strong activity against RSV (Fig.13).
9. Example: Hybrid polypeptides, obtained on the basis of luteinizing hormone
This example describes the hybrid proteins, obtained on the basis of luteinizing hormone (LH) and with improved pharmacokinetic properties. The methods described above, was synthesized and purified following hybrid LH-peptides: a core peptide T (Ac-QHWSYGLRPG-NH2and the hybrid polypeptide C (Ac-WQEWEQKIQHWSYGLRPGWASLWEWF-NH2), which contains the amino acid sequence of the crustal polypeptide T attached to the peptide-amplifier on amino - and carbonsilicon. As shown in Fig.12A and 12B, a hybrid peptide T had a significantly longer half-life compared to crustal peptide T, who lacked the sequence of the peptide amplifier.
10. Example: Pharmacology hybrid polypeptide T
T shown in Fig.13 is a hybrid polypeptide containing the sequence of peptide-amplifier attached to korovou the polypeptide, policyportal T has improved pharmacokinetic properties and has a strong in vitro, as well as in vivo activity against HIV-1. In the Example below describes pharmacological properties T as in rodents and primates, used as a model.
10.1. Materials and methods
10.1.1. A single dose of rodents
T was administered as a single dose to rats albino Sprague-Dawley by continuous subcutaneous infusion (SCI), subcutaneous injection (SC) or intravenous (i.v.). Each treated group consisted of nine rats of the same sex per group. These groups were injected with sterile preparations powdered medicinal substance C at a dose of 0.5, 2,0 or 6.5 mg/kg via CSI. One group was given 50 mm carbonate-bicarbonate, pH 8.5, as a control. These peptides were administered within 12 hours after polyvinylchloride/polyethylene catheter is surgically implanted under the skin in the back of the neck. Two groups were administered a single dose T, component 1.2 or 1.5 mg/kg by subcutaneous injection in vnutrimatocny region. Two groups were administered a single dose T, part 1.5, or 5 mg/kg by intravenous injection. The actual milligramme number T was calculated based on the content of the peptides that were identified for the introduction of each party.
The final stages and is, receiving data, clinical laboratory parameters, body weight and autopsy. Blood samples were taken by the method of the scattered sample within 12 hours from three rats of each sex per group at intervals: 0,5, 1, 2, 4, 6, 8, 9, and 12 hours after dose. The samples were assessed by using analysis of PcAb ECLIA (Blackburn, G. et al., 1991, Clin. Chem. 37:1534-1539; Deaver, D. 1995, Nature 377:758).
For analysis on the pharmacokinetic properties T in plasma and lymph in rats T prepared in the form of a sterile solution in bicarbonate buffer and injected in a single dose by intravenous bolus injection in the lateral tail vein at a dose of 20 µg/kg of The animal took the blood inserted through the jugular catheter. Samples were collected immediately after taking a dose after 5, 15 and 30 min and 1, 2, 4 and 6 hours after administration of the drug. For analysis of lymphatic fluid samples were taken immediately before the dose and every 20 minutes during the first six hours after a dose. Lymph fluid is collected through a catheter directly into the lymphatic thoracic duct cells, as described previously (Kirkpatrick & Silver, 1970, The Journal of Surgical Research 10:147-158). The concentration of T1249 in plasma and lymph fluid was determined using a camp shall maintain a single dose of primates
Sterile preparations powdered medicinal substance T1249 was introduced abacadabra apes in the form of a single dose via subcutaneous (s.c.), intramuscular (i.m.) or intravenous (i.v.) the injection. In subsequent cross-experiment, one group of animals consisting of two animals of each sex, were administered a single bolus dose T intravenous (0.8 mg/kg) intramuscularly (0.8 mg/kg) or subcutaneous (of 0.4, 0.8 and 1.6 mg/kg). Every day dose was accompanied by leaching period for at least three days. Just before the introduction of liofilizovannye T restored in sterile phosphate buffered saline pH 7.4. Real milligramme number of test drug was calculated based on the content of the peptide that has been defined for this input party.
The final stages of the analysis included an examination of the cells, a physical examination and determination of body mass. For i.v.-phase of this study blood samples were collected in heparinized tubes at the following time periods: immediately after a dose and through 0,25, 0,5, 1,5, 3, 6, 12 and 24 hours after dose. For i.m.- and s.c.-phases of this study, blood samples from each animal was collected in heparinised precisie one hour after collection and rapid freezing in liquid nitrogen. The samples were evaluated using ELISA PcAb (Blackburn, G. et al., 1991, Clin. Chem. 37:1534-1539; Deaver, D. 1995, Nature 377:758).
10.1.3. Interim pharmacokinetic studies
Six males abacadabra monkeys randomly divided into three groups of two animals each. All doses T in the form of a bolus was administered by subcutaneous injection. This study was divided into two stages. In stage 1 animals in groups 1, 2 and 3 were administered sterile drug powdered drug substance T (i.e. powdered T dissolved in carbonate-bicarbonate, pH 8.5) twice a day for four consecutive days (days research 1-4) at doses of 0.2, 0.6 and 2.0 mg/kg/dose, respectively. Stage 1 and Stage 2 were divided by the ten-day period of leaching. In Stage 2 animals in groups 1, 2 and 3 were administered sterile preparation of the finished composition T (i.e., in aqueous solution, pH 6.5 + 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 analyses were collected in the days of studies 1 and 15 for the evaluation of pharmacokinetic parameters after administration of single doses and days of study 4 and 18 to assess stationary pharmacokinetic parameters in ,5, 3,0, 4,0, 6,0, of 8.0 and 12.0 hours after administration of the dose. During Stages 1 and 2, the animals were continuously observed for clinical signs and changes in body weight.
10.2.1. Pharmacokinetics T introduced rats
A rat model was used to conduct a preliminary evaluation of the pharmacokinetics T in plasma and its distribution. Animals in all dose groups was not observed changes in body mass, data, physical examination, Haematology and clinical biochemistry data or macroscopic pathology associated with the introduction of T.
In rats, which were injected T by continuous subcutaneous infusion (c.s.i.), there is a constant concentration of peptides in the plasma of four hours after their introduction. As a constant plasma concentration (Cpss), and calculated the area under the curve (AUC) according to plasma concentration against time was directly proportional to the injected dose, indicating that T detects linear pharmacokinetics within the range of tested doses ranging from 0.5 to 6.5 mg/kg Calculated pharmacokinetic parameters and curve the concentration in the plasma from the time when S. s.i.-the introduction is presented in Table 9 and in Fig.16A, respectively.
esterwegen doses. In contrast, the introduction T by s.with.-injection was not given a dose-dependent pharmacokinetics within the studied doses. The calculated pharmacokinetic parameters and curve plasma concentration from time to s.c.-, and i.v.-introduction T presented in Table 10 and Fig.16B, respectively.
Was determined bioavailability T, administered to rats by subcutaneous injection compared with intravenous administration. The results are shown below in Table 11. At low dose (1.2 mg/kg) T found relative bioavailability (FR) constituting 73% for subcutaneous injection. Relative bioavailability T was 30% when the concentration C with the introduction of high-dose (15 mg/kg) administration was higher than the concentration that 90 % (IC90) inhibits HIV infection within 12 hours of the study at all tested doses.
Kinetic data for the concentration C in plasma and lymph illustrated in Fig.16C and systematized in the following Table 12. T quickly penetrated into the lymphatic system and balanced reservoir of drug substance in plasma within approx the only means in plasma and lymph were comparable within three hours after a dose at four of the five animals. One animal had consistently lower concentrations C in the lymph than other animals, however, the profile of elimination of the drug in the lymph of this animal did not differ from the profile of other members of this group. Comparison of half-life (t1/2) at the phase of elimination in plasma and lymph suggests that the transport T between these two compartments is a process regulated by diffusion. Three hours was observed the second, more rapid phase of elimination of lymphatic system. This difference may be caused by a specific mechanism (for example, due to redistribution or accelerated destruction of the peptide in the lymph) or other factors. Concentration C in the lymph fluid through 6 hours after injection exceeded the IC50for viral infectivity standard laboratory strains and primary clinical isolates of HIV-1.
It was also evaluated the degree of penetration of T in cerebrospinal fluid (CSF). Concentration C were below the limits of detection (LOD; 2,0 ng T/ml CSF) in all time intervals of the measurement, indicating that T does not penetrate into the Central nervous system after administration of a single dose.
10.2.2. kinetic parameters associated with parenteral introduction T, as models were used primates. Concentration T plasma in excess of 6.0 µg/ml and achieved by all the ways and quantitative levels (i.e. levels in excess of 0.5 μg/ml), were detected 24 hours after the s.c.- and i.v.-introduction. The breeding t1/2was comparable in all the ways (5.4 hours 4.8 hours and 5.6 for i hour.v.-, s.c.- and i.m.-introduction respectively). Concentration C in the plasma, which exceeded the value of the IC50for laboratory strains and clinical isolates of HIV-1, was observed in all periods of time measurements during 24-hour sampling.
Comparison of data obtained with parenteral administration of 0.8 mg/kg T for all routes of administration (s.c. i.v. and i.m.), presented on Fig.17A. In Fig.15V illustrated by comparison of data obtained in the result's.with.-injections at three different doses T (0.4 mg/kg, 0.8 mg/kg and 1.6 mg/kg). The insert of Fig.17B is a curve calculated AUC depending on the input dose.
T detects linear pharmacokinetics in abacadabra monkeys after it's.with.-introduction in the intervals of the injected dose, which indicates that at these intervals of missing doses(are) the mechanism(s) of saturation of the Kli is cauldrons below in Table 13. Comparison of the AUC values of plasma showed that, compared with intravenous administration, the bioavailability T with intramuscular injection of approximately 64% and percutaneously - 92%.
10.2.3. Interim pharmacokinetic studies
Interim pharmacokinetic study was performed to compare the pharmacokinetic profiles of plasma for powdered drugs T used in the clinical trials described above, with the profiles of the finished composition C, which was introduced a specific individual or patient, for example, for the treatment of HIV infection. This research was conducted with one-way cross-comparison of parallel groups that have introduced three levels of doses of powdered medicinal substance T and three-level doses of the finished product. Pharmacokinetics in plasma was assessed after administration of a single dose and after reaching steady state.
Introduction T by subcutaneous injection resulted in measurable levels of peptides for all dose groups. Curves in plasma concentrations with time were strictly parallel for all dose groups paleo substances T, and for the finished medicinal product T. In addition, the values of AUC(0-12 h)varied in direct proportion to the level of dose for both drugs. The calculated values of the AUC(0-12 h)for the finished medicinal product ranged from 43% to 80% values for AUC(0-12 h)calculated after administration of a single dose of medicinal substance, and from 36% to 71% at steady state.
Powdered medicinal substance T and finished medicinal product showed similar pharmacokinetic profiles in abacadabra monkeys after subcutaneous bolus injection at test level and volume doses. A direct comparison of the curves in plasma concentrations with time obtained in the present study, and the shapes of the curves obtained from previous studies conducted on abacadabra monkeys, gave reason to assume that there is a depot effect with the introduction of125I-T1249 by subcutaneous injection. This was confirmed by the increase in time, which reaches a maximum concentration in plasma (tmax), and t1/2.
These results showed that the drug powdered drug substance used in this pharmacological progabide after the introduction of the finished product. These observations showed that the introduction of T in clinical conditions, eventually leading to effects T on all patients.
Scope of the present invention is not limited to specific variants of its implementation described in this application and is presented only to illustrate certain aspects of the invention, and therefore functionally equivalent methods and components are also included in the scope of the present invention. Indeed, in addition to the modifications illustrated and described above, in the present invention can be introduced and various other modifications, which are obvious from the foregoing description and accompanying drawings. Such modifications should not go beyond the scope of the following claims.
ADDITION TO DESCRIPTION
The present invention relates to crustal polypeptides containing the following amino acid sequence:
DEFDESISQVNEKIEESLAFIRKSDEL; or QHWSYGLRPG.
In a preferred embodiment, a core polypeptide contains the amino acid sequence TALLEQAQIQQEKNEYELQKLDK.
Formula Isabeau the polypeptide, the sequence of peptide-amplifier comes from the consensus sequence of the envelope protein gp41 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, XWXXXW, XWXWXXX, XWXWXX, XWXW, WXWXXXW or XWXXXW, while the core peptide comprises a biologically active peptide, which has antiretroviral activity, growth factor, cytokine, the differentiating factor, interleukin, interferon, colony stimulating factor, hormone or angiogenic factor, and a hybrid polypeptide has improved pharmacokinetic properties compared with crustal polypeptide.
2. The hybrid polypeptide under item 1, characterized in that the sequence of peptide-amplifier contains WXXWXXXI, WXXXWXWX, IXXXWXXW or XWXWXXX.
3. The hybrid polypeptide under item 1, characterized in that the sequence of peptide-amplifier contains WQEWEQKI or WASLWEWF.
4. The hybrid polypeptide under item 1, characterized in that the sequence of peptide-amplifier connected to aminobenzo crustal polypeptide.
5. The hybrid polypeptide under item 4, further containing a sequence of peptide-amplifier connected to carboxylic crustal polypeptide.
6. The hybrid polypeptide under item 1, wherein TEI polypeptide under item 1, characterized in that the crustal polypeptide is a therapeutic agent.
8. The hybrid polypeptide under item 1, characterized in that the sequence of the peptide amplifier is about 4-30 amino acids in length.
9. The hybrid polypeptide under item 1, wherein the core polypeptide has the following amino acid sequence:
10. The hybrid polypeptide under item 9, characterized in that the sequence of peptide-amplifier connected to aminobenzo crustal polypeptide.
11. The hybrid polypeptide under item 10, further containing a sequence of peptide-amplifier connected to carboxylic crustal polypeptide.
12. The hybrid polypeptide under item 9, characterized in that after the d on p. 9, characterized in that the sequence of peptide-amplifier contains WQEWEQKI or WASLWEWF.
14. The hybrid polypeptide under item 9, characterized in that it contains the amino acid sequence of
15. The hybrid polypeptide under item 14, optionally containing aminobenzene acetyl group and carboxykinase amide group.
16. The hybrid polypeptide under item 10, wherein the core polypeptide has the following amino acid sequence TALLEQAQIQQEKNEYELQKLDK.
17. The hybrid polypeptide under item 16, further containing a sequence of peptide-amplifier connected to carboxylic crustal polypeptide.
18. The hybrid polypeptide under item 16 or 17, further containing aminobenzene acetyl group and carboxykinase amide group.
19. The hybrid polypeptide under item 13, wherein the core polypeptide has the following amino acid sequence: TALLEQAQIQQEKNEYELQKLDK.
20. The hybrid polypeptide under item 19, further containing aminobenzene acetyl group and carboxykinase amide group.
21. The hybrid polypeptide under item 14, characterized in that it contains the following amino acid sequence: WQEWEQKITALLEQAQIQQEK xianzu amide group.
23. The hybrid polypeptide under item 12, wherein the core polypeptide has the following amino acid sequence: TALLEQAQIQQEKNEYELQKLDK.
24. The hybrid polypeptide under item 23, further containing a sequence of peptide-amplifier connected to aminobenzo crustal polypeptide.
25. The hybrid polypeptide under item 23 or 24, further containing aminobenzene acetyl group and carboxykinase amide group.
26. The hybrid polypeptide under item 1, used for the manufacture of a medicinal product for the treatment of viral infections.
27. The hybrid polypeptide under item 9, used for the manufacture of a medicinal product for the treatment of viral infections.
28. The hybrid polypeptide under item 21, used for the manufacture of a medicinal product for treatment of viral infections.
29. A core polypeptide containing
30. A core polypeptide according to p. 29, additionally containing aminobenzene acetyl group and carboxykinase amide group.
31. A core polypeptide according to p. 29, containing TALLEQAQIQQEKNEYELQKLDK.
32. A core polypeptide on p. 31, optionally containing aminobenzene acetyl group and carboxykinase amide group.
33. A core polypeptide according to p. 29, used for the manufacture of a medicinal product for the treatment of viral infections.
34. A core polypeptide on p. 31 used for the manufacture of a medicinal product for the treatment of viral infections.
35. Method for improving the pharmacokinetic properties of the crustal polypeptide comprising the sequence accession peptide-amplifier to korovou the polypeptide with the formation of the hybrid polypeptide, so that its introduction into a living system, the hybrid polypeptide possessed improved pharmacokinetic properties compared with the pharmacokinetic properties of the crustal polypeptide, the sequence of peptide-amplifier comes from the consensus sequence of the envelope protein gp41 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, XWXXXW, XWXWXXX, XWXWXX, XWXW, WXWXXXW or XWXXXW, while cows the growth cytokine, a differentiation factor, interleukin, interferon, cholinesterase factor, hormone or angiogenic factor.
36. The method according to p. 35, characterized in that the sequence of the peptide amplifier is about 4-30 amino acids in length.
37. The method according to p. 35, wherein the core polypeptide has the following amino acid sequence
38. The method according to p. 35, wherein the core polypeptide contains TALLEQAQIQQEKNEYELOKLDK.
or its pharmaceutically acceptable salt, values radicals cm
< / BR>stimulating migration of neutrophils
where R1is benzyl or tert-butyl; R2not equal to R1and is selected from the group of tert-butyl; benzyl; 4-methoxybenzyl; 4-nitrobenzyl; diphenylmethyl; 2,2,2-trichloroethyl; 2,2,2-trichloro-1,1-dimethylethyl; allyl; 9-fluorenylmethyl; carboxamidine; substituted 2-sulfonylated type AND-SO2-CH2-CH2- where a is substituted or unsubstituted phenyl or benzyl; R3is a hydrogen atom or a urethane protective group of the B1O-CO-, where1not equal to R1and can take values tert-butyl, benzyl, 4-methoxybenzyl, 9-fluorenylmethyl, 2-(4-nitrophenyloctyl)ethyl; or is a peptidyl containing from one to three amino acid residues; and the peptides, where R3- peptidyl structure E-Z-Y-X-, in which E is a hydrogen atom or a urethane protective group IN2O-CO-, where2not equal to R1and can take values tert-butyl; benzyl; 4-methoxybenzyl; 9-fluorenylmethyl; 2-(4-nitrophen is IN3O-CO-, in which3= R1attached to the omega-amino group; Z can take values Phe or D-Phe