Peptide inhibitors of toxins derivatives of ll-37

FIELD: chemistry.

SUBSTANCE: invention concerns peptide compounds representing an amino acid sequence X1KEFX2RIVX3RIKX4FLRX5LVX6, where X1 is N-end segment which is IG; X2 is K or E; X3 is Q or E; X4 is D or R; X5 is N or E; X6 is C-end segment, which is a sequence selected out of PRTE or RPLR; where N-end segment is acetylated and/or C-end segment is amidated; with affinity to toxins, particularly to bacterial toxins, such as lipopolysaccharide or lipoteichoic acid. These compounds can inhibit or neutralise toxins. Invention also concerns pharmaceutical compositions and application of the claimed compounds in prevention or treatment of diseases or states caused by fungi or bacterial infection.

EFFECT: obtaining compounds for prevention or treatment of diseases or states caused by fungi or bacterial infection.

12 cl, 4 tbl, 4 ex

 

Description

The technical field relates to inventions

The invention relates to compounds that have an affinity for toxins, in particular to fungal and bacterial toxins such as lipopolysaccharide (LPS) or lipoteichoic acid (LTA), and which can inhibit or neutralize these toxins. In addition, the present invention relates to methods of producing such compounds and their therapeutic and diagnostic use, compositions comprising these compounds, genetic material, their codereuse, and methods of use thereof.

Prior art

Modern pharmacotherapy has made great strides in the fight against microbial and, in particular, bacterial infections, which was one of the main causes of premature death until the middle of the last century. However, in recent times there had been concern about the widespread use of highly effective antibiotics due to the constant increase in the resistance of bacteria. Indeed, over the last 25 years, resistance to antibiotics, in particular, multiple resistance to a wide range of antibiotic substances has increased in almost each of the investigated species of bacteria. Now suppose that the antibacterial agents are the most advanced type,which are not susceptible to traditional mechanisms of resistance, are compounds that are used as tools for the selection of mutants that are resistant to multiple drugs.

Based on this conclusion, the experts recommend the use of antibiotics is much more limited than previously, both in agriculture and in the treatment of the person. For example, minor illnesses - especially those who usually do not even caused by bacteria, such as the common cold, should not be treated with antibiotics, which should rather be reserved for more serious diseases. Moreover, for the treatment of bacterial infections it is necessary to develop new connections with different types of pharmacological activity, preferably with some activity which is not dependent on bacterial resistance to common antibiotics.

One of the diseases in which the widespread use of antibiotics has caused many disputes, is otitis media in its acute or chronic condition. It was shown that the number of patients with otitis media (OME), i.e. a kind of otitis media characterized by the presence of fluid in the middle ear without signs of acute infection, increased sharply after the appointment of antibiotic therapy in case of early acute otitis media (AOM), which suggests that the mi itself antibiotics play a role in OME (Lim et al., Laryngoscope 92, 278-286, 1982). I believe that such antibiotics as penicillin, influence the development of local immune responses, such as the development of the local IgM in the middle ear (Howie et al., Ann. Otol. Rhinol. Laryngol.,85Suppl. 25, 18-19, 1976). Another disadvantage of antibiotics is that bacteria are killed, but their toxins remain active.

It has been suggested that to treat these and other diseases caused by bacterial or fungal infection, it may be advantageous to use compounds that do not kill the microorganisms or bacteria themselves, but rather neutralize their toxins and provide natural protection mechanisms of the host to control the spread of infection (Nell, The Role of Endotoxin in the Pathogenesis of Otitis Media with Effusion, PhD Thesis, Leiden, 1999). At the same time, this strategy would support rapid recovery of functions of the weakened mucous membranes.

The main role among microbial toxins, such as fungal toxins and, in particular, bacterial toxins involved in a large number of infectious diseases such as otitis media, play endotoxins, group of lipopolysaccharides (LPS)found in the cell wall of gram-negative bacteria, consisting of a polysaccharide, United with highly toxic lipid a fragment, a lipid A. One of the newest therapeutic approaches to the treatment of OME is in the introduction the Institute of substances, which neutralize endotoxin or LPS (Nell, ibid.).

Currently, there are known various compounds capable of neutralizing endotoxin or LPS. For example, were developed several antibodies against endotoxins, such as NA-1A and E5, human, and mouse monoclonal IgM antibody, respectively. It was shown that these antibodies improve survival rate of patients with certain serious diseases such as septic shock (Ziegler et al., New Engl. J. Med.324, 429-436, 1991). However, a conclusion was made about their poor activity and specificity.

Another group of substances active against endotoxins, produced from endogenous human protein, called increasing the permeability of a bactericidal protein (BPI), which accumulates in azurophilic granules of neutrophils (Gazzano-three-betet. al., Infection and Immunity60:11, 4754-4761, 1992). BPI, which is a highly cationic protein, not only neutralizes free endotoxins, but also inhibits the growth or kills the bacterial cells themselves by increasing the permeability of their outer membranes. BPI is a potent natural antibiotic induced by the presence of LPS and some other triggering mechanisms, including tumor necrosis factor (TNF). However, a large part of its activity is associated with the synthetic is youdemi his immune cells, i.e. polymorphonuclear macrophages.

I received several recombinant proteins derived BPI, such as rBPI23(Kohn et al., 1993) and rBPI21(Horwitz et al., 1996), which largely represent the N-terminal parts BPI with molecular mass of 23 and 21 kDa, respectively. The use of BPI and compounds derived BPI in the treatment of OME were, for example, described in WO-A-00/71149.

Another class of natural compounds with antimicrobial activity are cathelicidin, class of peptides produced by cells of the respiratory epithelium, alveolar macrophages and other tissues. In their natural forms of these compounds are linear, α-spiral, not containing cysteine peptides or proteins. Cathelicidin are cationic and include highly conserved signal sequence and proregion, katelin. However, their C-terminal domain encoding the Mature peptide, shows considerable heterogeneity. These peptides can have from 12 to 80 amino acids.

The most promising human Catalina is 18 kDa cationic antimicrobial protein, SER. 37 C-terminal amino acids SER, i.e. peptide LL-37 is a domain responsible for the high affinity and neutralizing capacity against LPS (Sawa et al., Antimicr. Agents Chemoter.42:12, 3269-3275, 1998). We developed and testrow is but slightly shorter peptides, obtained from SAR or LL-37, such as disclosed Sawa (Sawa et al., ibid), Gustmann (Gustmann et al., Biophys. J.80, 2935-2945, 2001) and in U.S. patent No. 6040291 and its European duplicate EP-A-0955312.

Moreover, as the references cited article Isao Nagaoka et al. in Clinical and Diagnostic Laboratory Immunology9(5) (2002) 972-982 and article Kirikae et al. in Infection and Immunity66(5) (1998), 1861-1868.

Nagaoka et al. describe the amino acid sequence of LL-37 and obtained from him an 18-Mer To15-V32while Kirikae et al. focus on the number of patents derived from SAR-18.

Short peptides derived from LL-37, in which are preserved the natural amino acid sequence, and in particular such peptides comprising amino acid sequence KEFKRIVQRIKDFLRNLV are, therefore, described in the prior art.

In General, for several reasons, as the main candidate therapeutic compounds preferred in comparison with proteins, such as SAR are relatively small peptides. First of all, they can be more easily optimized, adapted and modified to preserve or strengthen their desired activity and specificity. Secondly, it is easier to obtain or synthesize and, thus, they are more available. Thirdly, they are more easily prepared in the form of drugs and to apply, while proteins are often the nest is stable and not bioavailable after parenteral administration.

The purpose of the invention

Despite the efforts of the earlier technology, there is a need for additional peptides and peptide compounds that possess LPS - and LTA-neutralizing activity and can serve as pharmaceutical or lead to the development of new pharmaceuticals for the treatment of diseases and conditions caused by bacteria, such as otitis media.

Moreover, there is a continuous need for such compounds without or with weak inflammatory activity, such as stimulation of cytokine production, proliferation of T cells, activation of extracellular signal-regulated kinase (ERK) or chemotaxis of neutrophils, all of the items listed are part of the known SAR-derived peptides.

One of the main goals of the invention is to provide new compounds which have affinity and neutralizing ability in relation to microbial toxins, in particular to fungal and bacterial toxins such as lipopolysaccharide (LPS) or lipoteichoic acid (LTA), but which at the same time have a reduced inflammatory activity.

An additional aim of the invention is the adaptation of known active amino acid sequences derived from LL-37 (and SAR-18), so that the functions of crods the VA and neutralization and inhibition are maintained at the same magnitude or even superior, while at the same time, the stability of these peptides optimized.

Another purpose is the provision of methods for obtaining such compounds, and therapeutic and diagnostic methods and compositions.

These and other objects of the present invention will become clear from the subsequent description.

Brief description of the invention

In the first aspect of the present invention provides compounds with affinity for lipopolysaccharides (LPS) or lipoteichoic acid (LTA). Compounds are peptide according to their chemical nature and include the amino acid sequence X1KEFX2RIVX3RIKX4FLRX5LVX6(here below also called Central (amino acid) sequence)in which X1represents the N-terminal segment of the sequence, X2is a or E, X3represents Q or E, X4is a D or R, X5represents N or E and X6represents the C-terminal segment; in which one or several amino acids in the Central sequence can be modified; in which the N-terminal segment is acetylated and/or C-terminal segment is emitirovannykh and/or amino acid sequence differs from the natural amino acid sequence of X KEFKRIVQRIKDFLRNLVX6.

In the second aspect, the invention provides methods for obtaining such compounds. Methods include chemical and enzymatic crosslinking of amino acid monomers or oligomers, to form compounds. The methods also include the expression of nucleic acids sequences encoding connection host cells using vectors for transfection of the host cell sequences of nucleic acids. The method of obtaining the compounds according to each of the preceding paragraphs, in which amino acid monomers, amino acid oligomers or mono - or oligomers of amino acid analogues or mimetics collect chemical or enzymatic crosslinking, which is carried out in the liquid phase and/or at the interface with functionalized solid phase.

In an additional aspect, the invention relates to the use of compounds according to the invention to obtain a pharmaceutical or diagnostic compositions, which are suitable for the diagnosis, prevention and/or treatment of diseases and conditions associated with or resulting from the presence of bacterial toxins, in particular LPS and LTA. These toxins can affect the body, even if by themselves infecting bacteria are no longer present in the body. Diagnostic compositions comprising the connection is possible under the present invention, can be usedin vivoorin vitro. Pharmaceutical compositions comprising compounds according to the invention will also normally contain one or more pharmaceutical carriers and/or excipients and will be adapted to be suitable for a particular method of administration, such as parenteral injection or infusion, topical application, such instillation, irrigation, injection or infusion; but also for inhalation, oral administration, nasal or injection through the mucous membranes or in any other suitable way. The composition can optionally contain agents that cause the targeted delivery of drugs, agents that increase the bioavailability, or active ingredients that are different from the compounds according to this invention and providing an immediate or modified release.

In an additional aspect, the invention relates to sequences of the nucleic acid encoding the peptide comprising the amino acid sequence, KEFX2RIVX3RIKX4FLRX5LV, in which X2is a or E, X3represents Q or E, X4is a D or R, X5represents N or E, where X2,X3,X4and X5do not represent simultaneously K, Q, D and N, respectively.

Additional speakers are the subjects of the invention will be formulated in the detailed description below and in the attached claims.

Detailed description of the invention

Compounds of the present invention are peptide compounds with affinity for lipopolysaccharides (LPS) or lipoteichoic acid (LTA). They include the Central amino acid sequence X1KEFX2RIVX3RIKX4FLRX5LVX6where X1represents the N-terminal segment of the sequence, X2is a or E, X3represents Q or E, X4is a D or R, X5represents N or E and X6represents the C-terminal segment; and where one or several amino acids in the Central sequence can be modified; where the N-terminal segment is acetylated and/or C-terminal segment is emitirovannykh and/or amino acid sequence differs from the natural amino acid sequence of X1KEFKRIVQRIKDFLRNLVX6.

In this description and the attached claims, the terms "N-terminal segment acetiminophen" have the following meaning. The N-terminal segment is protected by reaction with a carboxylic acid to obtain amide associated with stabilizing or protecting group. For example, the peptide may be introduced into the reaction with fumaric acid to obtain forestablishing peptide; with acetic acid to obtain azeti the protected peptide. Moreover, the peptide can be introduced into the reaction with propionic acid or other organic acids having up to 6 carbon atoms, and even up to 10 carbon atoms in the carbohydrate portion R. In these organic acids carbohydrate group R having up to 10 carbon atoms, can be unbranched or branched, or cyclic and/or contain one or more nancysinatra. Furthermore, the alkyl chain may be substituted, for example, hydroxyl group, halogen, amino, mercapto or sulfoxide groups. Therefore, the N-terminal segment of the next group can be presented: -C(O)-R'. Alternatively, instead of the reaction with the carboxylic acid, the reaction can also be carried out with sulfonic acid to obtain the corresponding sulfa communication. Therefore, in the N-terminal segment may contain the group-SO2-R. Alternatively, these terms also encompass alkylation and dialkylamino, so that in the N-terminal segment may contain secondary or tertiary amino group-N(R)1or-N(R)2in which each R has the above value.

In yet another embodiment, "acetylation" encompasses the reaction of the peptide with the isocyanate or isothiocyanato, in this case a urea or thiourea: R-N-C(O)- or R-N-C(S)-, respectively, where R is defined above.

the conclusion, The N-terminal segment may be protected by a blocking group that is stable to acid, where the group is usually injected in the course of peptide synthesis, but in this case it is not removed. Well-known blocking groups are Fmocand Z-group.

As for the meaning of the terms "where the C-terminal segment amitirova" noted the following. The term "amidation" means that HE usually present as of late, replaced by a group X, where X represents (i) a group-NY2where Y independently represents H or R, where R is the same as described above, or two groups Y together can form a cyclic fragment together with the N to which they are attached; (ii) the group-OR where R is, as described above, or (iii) a group-R. Peptide amides are preferred because they have the highest stability.

It was found that the peptide compounds of the present invention are optimized stability compared to natural amino acid sequence, are excluded from paragraph 1 of the claims.

Peptide compounds are peptides, such as oligo - or polypeptides, proteins or compounds obtained from peptides. In addition to the peptides, peptide compounds also include peptide analogs, derivatives, peptides, modified peptides and pertinacity. A common feature of peptide compounds is that they have amino acid sequences. More precisely, the peptides defined as amides, which can be obtained from two or more amino acids by combination of the amino group of one acid with the carboxyl group of another (Merriam Webster Medical Dictionary 2001). Peptide compound, in contrast, can also refer to a peptide structure within the molecule. Typically, peptides composed of natural (L)-α-amino acids, particularly alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), Proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

Analogs or functional equivalents of peptides are peptide molecules with the same activity and, in particular, the same affinity to microbial or features such as bacterial toxins, but not necessarily quantitatively, and can be, for example, modified peptides, peptide, peptide analogs, or peptidomimetics.

Modified peptides are molecules, peptides derived from the introduction of substituents Il the functional groups, which in General are not present in natural amino acids. This term also includes compounds that are produced by reaction of peptides with molecules from other chemical classes, regardless of whether there are these molecules in nature or not. For example, phosphorylated, from sulphonated and biotinylated peptides, glycoproteins and lipoproteins are often found in nature, while peptides modified with polyethylene glycol are examples of chemically modified peptides, which were designed to change some but not all properties of peptides.

Peptide, as peptides are peptide compounds. They also are usually inorganic salts of two or more amino acids. However, they often do not get directly from natural amino acids, but rather of different types of chemically synthesized L and/or D-amino acids.

Peptidomimetics, in their broadest sense, are compounds whose functional structure is more or less similar to the peptide, but which may also contain in its main chain ones due or D-amino acids. Generally, peptidomimetics are used as substitutes for natural peptides for interaction with receptors and enzymes (Pharmaceutical Biotechnology, Ed. D.J.A. Crommelin and R.D. Sindelar, Harwood Academic Publisher, 1997, p. 138). Pseudopath the water, the class of peptidomimetics are compounds containing isosteric amide bond instead of the amide linkages (ibid, pp. 137-140).

Compounds according to the invention also include salts of the peptides or functional equivalents, such as pharmaceutically acceptable additive salts of acids or bases and adducts. They also include multimeric peptides or functional equivalents.

Compounds according to the invention have an affinity for at least one toxin and, in particular, to a bacterial toxin. In particular, it was shown that the concentration of the peptides of about 1 micromoles demonstrate a significant (more than 50%) reduction in LPS/LTA activityin vitro. In fact, the term "affinity"as used herein, refers to the activity of the peptides used in the analyses. In these analyses, the preferred activity of the peptides was measured from low micromolar to high nanomolar range. Rather, the compounds according to the invention generally have submillimolar activity, the preferred compounds showing higher affinity, for example, with low micromolar or nanomolar activity. In a large number of infectious diseases of bacterial toxins, such as the class of lipopolysaccharides (LPS), in the case of gram-negative bacteria, and or lipoteichoic acid LTA), in the case of gram-positive bacteria, are associated with manifestations of the disease. These toxins can cause significant inflammatory reaction. For example, in infections of the upper respiratory tract inflammation can cause damage to the mucosa of the middle ear or sinuses that occurs due to the deterioration of the mucociliary clearance system (MCS), which represents one of the key protective systems of the upper respiratory tract. Compounds according to the invention is also particularly suitable for the treatment of intestinal infections. Affinity for fungal or bacterial toxins is a necessary condition for the ability to neutralize and, preferably, the compounds according to the invention not only associated with LPS and other toxins, but also has the ability to neutralize, inhibit toxins or other means to mitigate the effects of these toxins.

The desired type of activity against bacterial toxins observed in the case of peptide compounds perform structural requirements as defined in claim 1 of the formula of the invention, according to which these compounds include amino acid sequence X1KEFX2RIVX3RIKX4FLRX5LVX6in which X1represents the N-terminal segment of the sequence, X2is a or E, X3represents Q or E, X 4is a D or R, X5represents N or E and X6represents the C-terminal segment. This basic motif is derived natural antimicrobial protein SAR, or peptide LL-37, which itself is a derivative from SAR.

As used in this description, the N-terminal segment is a group, an atom or a sequence representing the N-terminal segment or domain connection, such as a structure that is attached to the end α-amino group of the primary sequence that does not form amide bond with this sequence. In the case of free α-amino group of the N-terminal site may merely represent a hydrogen atom; or it may consist of chemical groups attached to the nitrogen atom limit α-amino group, such as acyl group. It can also be a large group, such as a sequence of two or more amino acids, or chemical structure that is not or does not consist of amino acids. The C-terminal segment is similarly defined.

Preferably the compounds of the present invention include more than 18 amino acids that define the Central motif. In one embodiment, the implementation of the N-terminal segment consists of the sequence of the two is more amino acids. Among the amino acids, I and G are suitable members of this sequence, and the preferred N-terminal fragment is IG.

In another embodiment of the invention the C-terminal segment also includes the amino acid sequence. The sequence may include 1, 2, 3, 4 or more than 4 amino acids. In one embodiment, the C-terminal segment consists of 4 amino acids. With the end of the indicated C-terminal segment, consisting of 4 amino acids, can be an E, as in the corresponding position in the peptide LL-37. However, this s-end can also be determined R. Amino acid, neighboring with respect to the C-terminal amino acid, can represent T as in LL-37, but may also represent a L. P and R are two other preferred sequence of 4 amino acids C-terminal segment in any of the two remaining positions. More preferably, the C-terminal segment are selected from sequences PRTE and RPLR.

In an additional embodiment, the N-terminal segment and the C-terminal segment are selected from the preferred described above, to obtain the peptide sequence with the total number of amino acids 24. Among the most preferred in the present compounds, peptides IGKEFKRIVQRIKDFLRNLVPRTE and IGKEFKRIVERIKRFLRELVRPLR, or as the peptides themselves, or in the form of options is qualified, or derivatives of peptides.

Preferred among the modifications are amidarone and/or acetylated peptides according to the above definitions. One of the provisions in which the amidation seems to be particularly favorable, is the end of the peptide. Acetylation, on the other hand, is preferably produced by the N-end amino acids. In one preferred in the present embodiments, the peptides IGKEFKRIVQRIKDFLRNLVPRTE and IGKEFKRIVERIKRFLRELVRPLR are simultaneously acetylated at the N end and amidarone on the C-end. Preliminary tests confirmed that these modifications have increased stability in the presence of ectopeptidases.

Connection, as a rule, can be obtained by methods known to obtain peptides and related compounds. Smaller compounds that contain only a few amino acids or similar parts and, preferably, not more than 30-50 units, can be obtained by means of chemical or enzymatic crosslinking, or when using the classical approach, in which the reactions take place in solution or in suspension, or by using a more modern solid-phase approach, in which the peptide collect fixed on a solid surface, such as a polymeric bead. Large compounds are usually synthesized using automated solid-phase peptide synthesizers.

Alternatively, the compound can be obtained by known methods of genetic engineering. This approach is particularly effective if the compound is a peptide or slightly modified peptide. For example, the DNA sequence encoding connection may be associated or combined with expressing vector capable of transfection of cells. At another stage of the method, host cell or the target cell transferout specified DNA due to the contact of cells with DNA associated with the vector under conditions where it is possible to transfection. In the next stage of the host cell or the target cell is cultivated under conditions in which expression of the connection. Subsequently, the connection can be selected. If the connection itself may not be encoded or expressed, but it is very similar to the peptide, which can encode and Express, the method can be used to obtain peptide, which seems to be a connection, followed by one or more stages in which the peptide modify the chemical or enzymatic methods, obtaining the connection.

For these purposes, use different types of vectors, such as viral vectors, lipoplex, polyplex, microspheres, nanospheres, dendrimers, deproteinisation the DNA peptide delivery system, lipids, particularly cationic lipids, or derived liposomes, polymeric vectors, in particular polymeric vectors derived from poly-polymers. Among the preferred viral vectors are presented retroviruses, adenoviruses, adrenosterone viruses, herpes simplex viruses and virosome. Preferred non-viral vectors include chitosan, SPLP, polymeric systems based on PLGA, polyethylenimine, polylysine, palifosfamide, poly(meth)acrylates, polyphosphazene; DOPE, DOTAP and DOTMA.

A somewhat more extensive reviews of methods that you can use to obtain the compounds according to the invention described in: W.F. Anderson, Nature392Supp., 30 April 1998, p. 25-30; Pharmaceutical Biotechnology, Ed. D.J.A. Crommelin and R.D. Sindelar, Harwood Academic Publishers, 1997, p. 53-70, 167-180, 123-152, 8-20; Protein Synthesis: Methods and Protocols, Ed. R. Martin, Humana Press, 1998, p.1-442; Solid-Phase Peptide Synthesis, ED. G.B. Fields, Academic Press, 1997, p. 1-780; Amino Acid and Peptide Synthesis, Oxford University Press, 1997, p. 1-89.

Salts of the peptides or functional equivalents get by known methods, which usually involve mixing the peptide or peptide or pharmaceutically acceptable acid with the formation of additive salts of acids or with pharmaceutically acceptable base with the formation of the additive salt of the base. Whether acid or a pharmaceutically acceptable base, can be easily installed by a specialist in the Anna area, taking into account how the connection will be used for the purpose. For example, not all acids and bases, which are acceptable for diagnostic compositionsin vitrocan be used for therapeutic compositions. Depending on the purpose of use of pharmaceutically acceptable acids include organic and inorganic acids, such as formic acid, acetic acid, propionic acid, lactic acid, glycolic acid, oxalic acid, pyruvic acid, succinic acid, maleic acid, malonic acid, cinnamic acid, sulfuric acid, hydrochloric acid, Hydrobromic acid, nitric acid, perchloric acid, phosphoric acid and Tiziana acid, and which form the ammonium salt with the free amino groups of peptides and functional equivalents. Pharmaceutically acceptable bases, which form a carboxylate salt with the free carboxyl groups of peptides and functional equivalents include ethylamine, methylamine, dimethylamine, triethylamine, Isopropylamine, Diisopropylamine and other mono-, di - and trialkylamines, as well as arylamine. Moreover, also covered pharmaceutically acceptable solvate, complexes or adducts, such as hydrates or aturity.

Some of predpochtitelnei peptides according to the present invention can be easily produced during or at the end of the synthesis. For example, if the peptide is synthesized using solid-phase method, acetylation by N-end can be performed at the end of the synthesis by the introduction into the reaction amino acid sequence that is still linked to the resin with acetic acid instead of another amino acid.

On the other hand, the amidation at the C-end can be when used in solid-phase peptide synthesis is a special type of resin, such as commercially available Tentagel S AM (ex, Rapp, Tübbingen, Germany). These resins include chemical "handle", which amidarone peptides are released during decomposition. These and other methods of modification of peptides known to any person skilled in the technical field.

An additional aspect of the invention relates to the use of peptide compounds. The compounds have affinity to microbial toxins and, in particular, to bacterial toxins such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Therefore, the compounds can be advantageously used in therapeutic and diagnostic purposes in diseases and conditions associated with the presence of these toxins. Binding capacity is, as a rule, lead to the neutralization of toxins, making connections can be viewed as antagonists or partial antagonists. Moreover, they can be used the Ana as targeting agents or ligands for other compounds, capable of neutralizing the toxins that can be specifically targeted to these toxins through covalent or non-covalent linkage with these compounds, or by covalent or non-covalent binding to the surface of the carrier of a medicinal product, such as a liposome, a nano - or microparticle, nano - or microcapsule, lipid complex or micelle.

When diagnosing connection can be used for qualitative or quantitative analysis of bacterial toxins present in physiological fluids, such as blood, plasma, serum, mucus lining the epithelium of the mucous membranes, such as the respiratory tract, or in liquids, the presence of which is associated with pathological conditions such as fluid is detected in the middle ear with exudative otitis media (OME). For such applications, the compounds can be included in diagnostic kits for usein vitroor diagnostic compositions that can be entered for the patient. For this application, an optional variant involves the binding of the compounds according to the invention with a chelating agent, which then forms a complex with isotopic label, find an acceptable control system.

In a preferred application, the compound is administered as the active medicinal substances for p is edatrexate or treatment of diseases or conditions, associated with fungal or bacterial infections or the presence of fungal or bacterial toxins in the body. As already noted, there are certain disadvantages and limitations of antibiotic therapy in acute or chronic infections, such as induction of resistance and selection of resistant varieties of bacteria, suppression of the natural defense systems of a patient, a violation of bacterial flora that inhabit the mucous membranes in vivo, the release of a large number of bacterial toxins, when you die, bacteria, etc. moreover, there may be illness and disease, in which the main reason is the presence of toxins and, in particular, bacterial toxins, but the presence of microorganisms, as in OME, when the local retention of toxins in the middle ear may have a significant contribution to the manifestations of the disease, even in the absence of symptoms of acute infection.

In all these cases, it may be desirable to treat the disease, not drugs antibiotics and substances that can neutralize bacterial toxins. For this purpose, the compounds according to the invention is particularly advantageous, because they show high binding and neutralizing activity against the most important microbial toxins such as lipopolysaccharide (LPS) in the case of gramatica the nutrient and bacteria lipoteichoic acid (LTA) in the case of gram-positive bacteria. Infections of the upper respiratory tract for the treatment of which the compounds according to the invention are particularly preferred, these waste products of bacteria can cause inflammation in the middle ear or sinuses and can cause damage to the mucous epithelium of the upper respiratory tract. Neutralization included toxins may allow to prevent, control or reduce the damage of the mucous membrane, including disorders of mucociliary system kerensa (MCS), and thus will strengthen the natural protective system. In such cases, including OME, in which bacterial toxins can be a major problem even in the absence of a significant number of live bacterial cells, therapy based on the introduction of the compounds according to the invention, for example, directly into the middle ear, can be a primary therapeutic approach. But also other respiratory tract infections such as acute or chronic sinusitis or acute or chronic otitis media, these compounds may be extremely suitable for restoring the normal functions of the mucous and their natural protective systems.

In a more General form of the compounds according to the invention are agents that are useful for the prevention and treatment of conditions caused by infectious bacteria, the key Streptococcus pneumoniae,Haemophilus influenzae,Moraxella catarrhalis,β-hemolytic Streptococcus group a,Staphylococcus aureusgram-negative enteric bacteria,Streptococcus pyogenes,Escherichia coli,gram-negative bacilliPseudomonas sp.

A particular advantage of the compounds of the present invention in comparison with natural proteins and peptides from which they derive, such as SAR and LL-37, is the low degree of their undesirable inflammatory activity. This activity is associated with various cellular processes, including proliferation, differentiation and expression of genes encoding Pro-inflammatory mediators, such as cytokines. Cytokines are direct mediators of inflammation and influence the development and direction of many immunological reactions. The imbalance in the production of cytokines is widely known as a critical factor in several painful conditions. When conditions such as exudative otitis media or sinusitis, this balance is distorted. The proliferation of T-cells in such conditions is not favorable, because it additionally stimulates the immune response, which is not monitored.

Thus, these compounds can be successfully used in the pharmaceutical compositions. According to the invention such farm is septicemia compositions are provided as are the connections. As used herein, the term "pharmaceutical composition" refers to therapeutic and diagnostic compositions, as well as to drugs and diagnostics, containing such compositions. Therapeutic compositions and drugs used to prevent or treat diseases and other conditions in mammals, the improvement which seems desirable. Diagnostics and diagnostic compositions used for diagnostirovanie such diseasesin vivoandin vitro.

Typically, such compositions comprise at least one compound according to the invention as active ingredient and at least one pharmaceutically acceptable carrier or excipient. Moreover, the composition of processed and prepared in such a way that it can enter the human or animal. As used in this description, the media or excipient represent any pharmaceutically acceptable substance or mixture of substances that do not have significant pharmaceutical activity, which can be used as a carrier or excipient, to obtain compounds in pharmaceutical dosage form, which is stable and suitable for introduction. Examples of pharmaceutically acceptable excipients known the s professionals and can be found in the monographs on the main Pharmacopoeia.

In one of the embodiments of the composition is formed and prepared for parenteral administration, instillation, or douching, preferably for intravascular injection, such as intravenous or intra-arterial, but also intramuscular, subcutaneous, intralesional, intraperitoneal, local, or other means of parenteral administration. In another preferred embodiment, the composition is injected directly on the damaged mucosa of the upper respiratory tract, for example, of the middle ear. The same principles that guide the design formulas of other drugs such techniques are used by specialists in this field for obtaining such compositions. For example, one of the requirements for pharmaceutical dosage forms for parenteral injection is sterile. Other requirements described in all major pharmacopoeias, such as in USP 24, in the monograph "General Requirements for Test and Assays. 1. Inhections", p. 1775-1777. To improve the stability of formulations for parenteral administration may be necessary to obtain a dry pharmaceutical dosage forms, which must be restored before the introduction. An example of such drug dosage forms are freeze-dried or lyophilized drugs. The composition and the finding may also contain a mucolytic solvent.

It may be desirable to introduce the connection according to the invention in the form of a parenteral pharmaceutical dosage forms with controlled release in order to avoid frequent injections and improve the efficiency and convenience of therapy. There are various ways to get depo-compositions. Prolonged release can be provided by use of solid implants, nanoparticles, nanocapsules, microparticles, microcapsules, emulsions, suspensions, oil solutions, liposomes or similar structures.

In the case of compositions, which must be administered topically to the affected mucous membranes may be useful to provide a composition having properties that provide long time local retention at the injection site, to improve the effectiveness of treatment. To achieve this goal in the composition can be included mucoadhesive excipients. Such functional excipients known to the person skilled in the technical field; they include polymers, such as polyacrylic acid and derivatives thereof, polymethacrylic acid and their derivatives, cellulose ethers, including hypromellose, carboxymethylcellulose, starches, chitosan, etc. are Suitable, or alternatively, the composition of the invention may also contain a mucolytic solvent. In particular, mucolytic races is Writely used to impact on the capacity of penetration of the peptide compounds according to the invention in mucus, for example, in the respiratory tract. Suitable solvents may include known mukoregulyatornym or mucolytic agents such as N-acetylcysteine, S-carboxymethylcysteine, Bromhexine, Ambroxol, Tnkase, erdosteine, saline, nicotein. Preferably use Bromhexine.

Additional excipients that are particularly useful for the preparation of dosage forms for parenteral administration, in the broadest sense, are solvents, co-solvents and liquid or semi-solid carriers, such as sterile water, ethanol, glycerin, propylene glycol, butanediol, fatty acids, short and srednezerny triglycerides, lecithin, polyoxyethylene derivatives of castor oil; substances for establishing osmotic pressure and pH, such as sugars, in particular glucose, sugar alcohols, especially beckons, sodium chloride, sodium carbonate, citric acid, acetate, phosphate, phosphoric acid, hydrochloric acid, sodium hydroxide, etc.; stabilizers, antioxidants and preservatives, such as ascorbic acid, sulfite or hydrosulfite sodium ethylenediaminetetraacetate, benzyl alcohol, etc., other excipients and auxiliary means for lyophilization, such as albumin, dextran etc.

In a similar manner can be advantageous centuries the presence of the compounds according to the invention in the form of pharmaceutical dosage forms for administration through mucosa. This method of administration is non-invasive and harmless in relation to the patient; at the same time, it usually leads to improved bioavailability of the compounds according to the invention compared with orally administered, especially if the connection is unstable in the fluids of the digestive system or if it is too large to be effectively absorbed in the intestine. Introduction through the mucous may, for example, using a nasal, transbukkalno, sublingual, gingival, or vaginal pharmaceutical dosage forms. These pharmaceutical dosage forms can be obtained well-known technological methods; they can be tailored to provide nasal drops or sprays, strips, films, patches, gels, ointments or pills. Preferably, the excipients used for pharmaceutical dosage forms for administration through mucosa, also include one or more substances, intended for mucoadhesive, thus increasing the contact time of drug dosage forms to the place of absorption and, thus, potentially increasing the duration of absorption.

Alternatively, the pharmaceutical composition may be intended for oral administration and appropriately made. Suitable oral drug dosage forms in luchot pills, hard capsules, soft capsules, powders, granules, pharmaceutical dosage forms, disintegrating in the oral cavity, syrups, drops, suspensions, effervescent tablets, chewable tablets, an oral film, lyophilized pharmaceutical dosage forms pharmaceutical dosage forms with prolonged release pharmaceutical dosage forms with controlled release. In one of the preferred embodiments, oral pharmaceutical dosage form is a solid medicinal dosage form with an enteric coating to protect the connection from the acidic and proteolytic environment of the stomach.

The composition can also be developed for intracolonic injection.

In an additional embodiment, the compound is administered through the lungs, while using nebulizer with a controlled dose of the aerosol generator, the aerosol spray or dry powder inhaler. Acceptable formulations can be manufactured using known methods or techniques. Also, in some cases, it may be feasible transcutaneous, rectal or intraocular administration.

It may be advantageous to use proactive introduction of drugs or directed methods for more effective delivery connection p of the invention. For example, if you choose aparentally route of administration, suitable pharmaceutical dosage form may include an agent that enhances the bioavailability, which can be any substance or mixture of substances that enhance the availability of a connection. This can be achieved, for example, the connection protection from decomposition, such as enzyme inhibitor or antioxidant. More preferably, the reinforcing agent enhances the bioavailability of the compound by increasing the permeability absorption barrier, which is usually the mucous membrane. Amplifiers permeability can operate using different mechanisms; some increase the fluidity of mucous membranes, while others open or expand the slotted contact between mucous cells. Others reduce the viscosity of mucus overlying the mucosal cells. Among the preferred amplifiers permeability are amphiphilic substances, such as derivatives holeva acids, phospholipids, ethanol, fatty acids, oleic acid, derivatives of fatty acids, ethylenediaminetetraacetate, carbomer, polycarbophil and chitosan.

The following examples serve to illustrate the invention without limiting its scope described in this description of the options for implementation.

Example 1: Obtaining connections

Amino acid sequences of the compounds represented:

R IGKEFKRIVQRIKDFLRNLVPRTE

RAS* IGKEFKRIVQRIKDFLRNLVPRTE

R IGKEFKRIVERIKRFLRELVRPLR

RAS* IGKEFKRIVERIKRFLRELVRPLR

* The index of the Speakers means that the peptide acetiminophen N-end and amitirova on the C-end.

Example 2: Neutralization of toxins

The compounds obtained according to example 1, were analyzed for their ability to neutralize bacteria the capacity toxin LPS breakdown of the lysate of amoebocytes of limulus (LAL) and the breakdown with whole blood (WB). LTA neutralization also measured breakdown with whole blood. Peptide LL-37 is also used as a positive control. The concentration of peptide, whereby neutralized 50% of LPS (50% inhibition)was used as a measure of the activity of the peptide. These values of concentrations were as shown in table 1. The differences between the compounds in each analysis were not statistically significant. In conclusion, the tested compounds according to the invention showed about the same degree antitoxic activity, as natural antimicrobial agent LL-37.

Table 1
Peptide50% inhibition of LTA (mg/ml), n=350% inhibition of LPS (µg/ml)
LAL-analysisWB analysisaverage
LL-371,6±0,51,3±0,2 (n=5)1,2±0,2 (n=3)1,3±0,2 (n=8)
P602,1±0,71,5±0,5 (n=5)1,4±0,1 (n=4)1,5±0,3 (n=9)
P60.42,0±1,31,7±0,6 (n=5)2,1±0,6 (n=2)1,8±0,6 (n=7)
P60.Ac2,1±0,11,8±0,8 (n=5)2,4±0,(n=2) 2,0±0,8 (n=7)
Table 1: the values for 50% inhibition of LTA (±standard deviation) in µg/ml and value of 50% inhibition of LPS (±standard deviation) in mcg/ml LL-37, P60, P60.4 and RAS. Inhibition of LTA experienced in the analysis with whole blood (WB). Inhibition of LPS tested in LAL-analysis, as well as in the analysis with whole blood. Synthetic peptides caused neutralization of the LTA, which did not differ significantly from LL-37. Neutralization of LPS caused by synthetic peptides, also did not differ significantly from LL-37. n=number of experiments.

Example 3: the Activation of immune cells through connections

The compounds obtained according to example 1, were tested on their therapeutically undesirable immunological activity using assays Elispot, proliferation of T cells, ERK activation and chemotaxis of neutrophils. Elispot analysis is used to determine the action of a drug, chemical substance or other compounds on the secretion of the cytokinein vitroso getting information about their possible modulatory activity on immune functionin vivo. The results are presented as the percentage of positive responses to IFN-gamma. ERK-(extracellular signal-regulated kinase-1/2 is part of a signaling pathway MAR-kinases is, which, as has been shown, included in various cellular processes, including proliferation, differentiation and expression of genes encoding Pro-inflammatory mediators, such as cytokines. Cytokines are direct mediators of inflammation and influence the development and direction of many immunological reactions. The imbalance in the production of cytokines is widely known as a critical factor in several painful conditions. When diseases such as exudative otitis media or sinusitis, this balance is distorted. The proliferation of T-cells in such conditions is not favorable, because it also stimulates the immune response, which is not controlled. Thus, it is desirable that the compounds according to the invention is not stimulated production of cytokines, proliferation of T-cells, ERK-activation and chemotaxis of neutrophils.

For the proliferation of T-cells 150000 peripheral mononuclear blood cells (PBMC) were cultured in the absence or in the presence of 10 μg/ml of the compounds for 5 days in 96-well round-bottom microplate (Costar Inc. Cambridge, MA) in a final volume of 150 μl of complete IMDM. As a positive control PBMC were cultured in the presence of 25 u/ml recombinant IL-2. In the last 20 hours of cultivation of PBMC pulsed noted [3H]thymidine (0.5 microcurie/well), after which it was measured by the inclusion of 3N when using liquid scintillation counting. To determine cytokines, T cells, IFN and IL-10 Elispot study, 1,5×106PBMC were cultured in 0.5 ml of complete IMDM in the absence or in the presence of different concentrations of synthetic peptide. As a positive control PBMC stimulated with 10 μg/ml of the mitogen Phytolacca American (PWM). After 48 hours of cultivation RVMS gathered careful washing of the wells warm IMDM to collect not adhering cells, which were washed in a large volume of IMDM. Then RWMS was placed in tablets, pre-coated with an antibody ELISA, and were cultured for 5 hours in IMDM, supplemented with 2% of the combined AB human serum at 37° WITH 5% CO2after which the tablets were shown according to the Protocol from the manufacturer (U-CyTech, Utrecht, The Netherlands). Spots were counted using Olympus microscope and analyzed using software Olympus Micro Image 4.0 (Paes Nederland, Zoeterwoude, The Netherlands). The final results are presented as the percentage of positive indexes stimulation (positive: >2).

Activation of ERK-1/2 was analyzed with the cells from the cell line mucoepidermoid tumors of the lung NCI-H292 (ATCC, Rockville, MD), which were cultured in 24 - or 6-hole tablets to be cultivated in RPMI1640 medium (Gibco, Grand Island, NY) supplemented with 2 mm L-glutamine (Bio Wittaker, Walkersville, MD), 20 u/ml of penicillin (Bio Wittaker), 200 μg/ml streptomycin (Bio-Wittaker) and 10% (vol./about.) amniotic calf serum inactivated by heating (Gibsco). After reaching close clusters, cells were cultured overnight in medium containing no serum. The cells are then stimulated for 15 minutes with these stimulating effects. Lysates of cells were obtained using lyse buffer (0,5%[vol./about.] Triton X-100, 0.1m Tris-HCl pH to 7.4, 100 mm NaCl, 1 mm MgCl2, 1 mm Na3VO4mini full of a cocktail of protease inhibitor [Boeringer Manheim, Roche, Basel, Switzerland]). The samples were subjected to SDS-PAGE on 10% gel based on glycine and separated proteins were transferred to polyvinylidenedifluoride (PVDF) membrane. Nonspecific binding sites were blocked with a mixture of PBS/0.05% Tween-20/1% casein. Replica incubated with polyclonal rabbit antibodies against phosphorylated ERK-1/2 (New England Biolabs, Beverly, MA) and secondary horseradish peroxidase conjugial with antibodies against rabbit IgG. For detection of immunoreactivity use advanced chemiluminescent system Western blotting detection (Amsterdam Pharmacia Biotech, Upsala, Sweden).

Chemotaxis of neutrophils was measured using neutrophils isolated from peripheral blood using Percoll density centrifugation (density: 1,082 g/ml). Cells are re-suspended at a concentration of 2.5×106to etoc/ml in the medium of chemotaxis (20 mm N-2-hydroxyethylpiperazine-N'-2-econsultancy acid (HEPES buffer), HEPES, 132 mm NaCl, 6 mm KCl, 1.2 mm KH2PO4, 1 mm MgSO4, 5.5 mm glucose, 0.1 mm CaCl2and 0.5 % (wt./about.) serum albumin [Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (CLB), Amsterdam, The Netherlands], diluted 1:1 serum-free RMPI. Chemotactic activity of the compounds was assessed using a modified method of Boyden of Camber. Briefly, 26 μl of stimulants, diluted with HEPES buffer, were added to the wells of the lower compartment and 50 μl of the suspension of neutrophils (2,5×106cells/ml) were added to the upper compartment. The compartments were separated by two filters: lower filter with pore size 0.45 µm (Millipore Products, Bedford, MA) and the top filter with a pore size of 8 μm (Sartorius Filter, San Francisco, CA). After incubation for 90 minutes at 37aboutWith upper filters were removed, fixed in a mixture of ethanol-butanol (80:20./about.) and stained with a solution of Weigert. To determine the chemotactic activity of neutrophils neutrophils were counted in six random fields at high magnification (×400) and calculated the percentage of neutrophils in the membrane compared with the positive control (10-8M N-formylmethyl-leucyl-phenylalanine (FMLP, Sigma).

The results are presented in table 2. In conclusion, the tested compounds according to the invention, and, in particular, R, caused a very low immune response, lower than the natural peptide LL-37. They showed the bottom of the eighth ERK activation and the practical absence of chemotaxis of neutrophils.

Table 2

Immunogenicity of connections
Connectionγ-IFN ElispotProliferation

T-cell
ERK activationChemotaxis (%)
P601/80/8-76±39
P60.Ac3/80/8±61±36
P60.43/80/8±0±0
RASnd0/8±24*
LL-37(control)4/84/8+84±17
*only one dimension

nd=not determined

Example 4:In vivoportability

Connection RAS was obtained according to example 1 and tested for its portabilityin vivo. More specifically, was evaluated by its ability to cause irritation of the eyes and skin of rabbits, while its ototoxicity was studied on the model of the Guinea pig. Moreover, its systemic toxicity was assessed after intravenous injection.

For experiments on eye irritation and skin of three rabbits were exposed to 0.5 ml of buffered fosfato the solution of peptide (2 mg/ml), applied to the shaved skin for 4 hours when using preoccupies dressing. The observations were carried out after 1, 24, 48 and 72 hours after exposure. Individual samples of 0.1 ml of phosphate buffered solution of peptide (2 mg/ml) (pH 7.5) were buried in one eye of each of three rabbits for the study of acute irritation/damage to eyes. The observations were carried out after 1, 24, 48 and 72 hours after instillation.

In the result there was no evidence of skin irritation. The instillation of a solution of the peptide in the eye resulted in redness of the conjunctiva, which completely disappeared within 20 hours after instillation.

Systemic toxicity RAS evaluated in toxicity studies with single and repeated doses in rats. The peptide was injected daily intravenously with increasing doses. At this stage determined the maximum tolerated dose (MTD). The toxicity of repeated doses has also studied at the stage MTD. At the stage of increasing doses of 9 rats were divided into three groups and they received of 0.4, 2, or 8 mg/kg/day for two days. Clinical signs were recorded twice daily on the day of dosing and one day after dosing, body weight was recorded before the first dose and after one day after dosing. On MTD stage 5 females and 5 male rats received 8 mg/kg/day for 5 successive days. Clinical symptoms were recorded two R is for a day on the days of dosing, body weight at day 1 and 6. Clinical laboratory studies were performed before the autopsy. Macroscopia conducted after the end of the MTD phase.

As a result, when the systematic studies of the dose increase was not observed in mortality. Moreover, in clinical symptoms and body weight were not observed clear deviations. During the MTD phase was not observed mortality and there were no clear conclusions related peptide in clinical symptoms, body weight, Hematology and clinical biochemical parameters and macroscopic study.

For the evaluation of ototoxicity used 9 healthy male Guinea pigs-albinos (500-1200 g) without external ear abnormalities. Animals were anestesiologi intraperitoneal injections of ketamine 40 mg/kg and rompun 10 mg/kg After conducting tests of hearing surgically opened auditory vesicle for applying a small piece of spongostan on the membrane window of the cochlea (RWM) and various solutions (approximately 10 μl) were injected on spongostan. The skin was sutured and then conducted additional testing of hearing.

Guinea pigs were divided into three groups, each consisting of two animals being treated and one control animal. The test and control compositions were introduced RWM right ear and the left ear was left without education is ODI. In group 1, two Guinea pigs received cisplatin (0,66 mg/ml in PBS) and one animal that received PBS as a control. The cisplatin ototoxicity known, served as a positive control in the test. Group 2 received the peptide (2 mg/ml) in phosphate buffer (pH 7.5) and group 3 received the peptide (2 mg/ml) in solution, comprising 7% of macrogol 10000 in isotonic sodium chloride, canned using 0.02% benzalkonium chloride and 0.1% Na2EDTA, buffered with phosphate at pH 5.5.

The acoustic response of the brainstem (ABR) were recorded using an automated system signal averaging (Tuker-Davis Technology, Aluha, FL, USA) before conducting drug and immediately after surgery and after 3, 7, 14, and 22 days. Guinea pigs were anestesiology and into the external ear canal were placed head-phone ear Bud. Subcutaneous electrodes were placed over the top (active) and on hearing the bubble on the same side (the reference electrode). Ground electrodes were placed over the neck muscles. ABR was recorded in shielded from electricity, double wall, shielded from the radio room in response to 10 MS tone parcel at 1kHz. The intensity of the stimulus was measured and expressed in dB. Threshold ABR was defined as the lowest intensity capable of causing playback is possible, visually detectable response. Threshold ABR after treatment compared with the threshold values ABR before processing.

As a result, the use of PBS in the window of the cochlea in group 1 resulted in the change of the threshold value -9 dB after 22 days after surgery, whereas cisplatin caused a change -49 -64 dB and dB, respectively (see table 3). In the second group, the use of phosphate buffer did not cause changes in the threshold values (see table 4). Phosphate buffer with 2 mg/ml of the peptide caused the change threshold -7 dB after 22 days after surgery. In one animal, it was impossible to open the auditory vesicle and the animal was excluded from the study. In the latter group the introduction of the peptide in the composition of the solution led to a very small change in the threshold values of 2 and 1 dB, respectively (see table 5).

Table 3

Ototoxicity of cisplatin (positive control)
Group 1Only PBSCisplatin in PBSCisplatin in PBS
Threshold (dB)Δ before surgeryThreshold (dB)Δ to chirurgic the ski intervention Threshold (dB)Δ before surgery
Before surgery865882
After surgery72-1456-281-1
3 days67-1926-3244-38
7 days76-1026-3237-45
14 days78-828-3023-59
22 days77-99-4918-64

Table 4

No ototoxicity RAS in phosphate buffer
Group 2Only PBSRAS in PBS
Threshold (dB)Δ before surgeryThreshold (dB)Δ before surgical intervention is TBA
Before surgery7772
After surgery78170-2
3 days74-370-2
7 days74-3720
14 days77060-12
22 days77065-7

1. Peptide compound with affinity to bacterial and fungal toxins, and in particular to the lipopolysaccharide (LPS) or lipoteichoic acid (LTA), which represents the amino acid sequence X1KEFX2RIVX3RIKX4FLRX5LVX6,

in which X1represents the N-terminal segment, representing IG;

X2is a or E;

X3represents Q or E;

X4is a D or R;

X5represents N or E;

X6represents the C-terminal segment, which is a sequence selected from PRTE or RPLR;

in which the N-terminal segment is etilirovannym and/or C-terminal segment is emitirovannykh.

2. The compound according to claim 1, consisting of a sequence of 24 amino acids or their derivatives, in which the sequence selected from IGKEFKRIVQRIKDFLRNLVPRTE and IGKEFKRIVERIKRFLRELVRPLR.

3. The compound according to claim 2, in which the N-terminal segment acetiminophen and C-terminal segment amitirova.

4. The use of compounds according to claim 1 for the diagnosis, prevention or therapy of a disease or condition involving or resulting from fungal or bacterial infection, or exposure to fungal or bacterial toxin, such as lipopolysaccharide (LPS) or lipoteichoic acid (LTA).

5. The use according to claim 4 for the treatment of a fungal or bacterial infection of the upper respiratory tract or respiratory system, or diseases that are their result, such as acute or chronic sinusitis, acute or chronic inflammation or exudative otitis media.

6. Pharmaceutical composition having LPS - and LTA-neutralizing activity, comprising a compound according to claim 1 and one or more pharmaceutically acceptable carriers or excipients.

7. The pharmaceutical composition according to claim 6, composed, produced and intended for parenteral administration, preferably for intravascular, intramuscular, subcutaneous or intralesional introduction.

8. The pharmaceutical composition according to claim 6, composed of otulana and designed for the local introduction of the mucosa of the affected area or tissue, such as in the form of leaching fluid, ear drops, nasal drops, spray, powder, aerosol, liquid, spray, gel, suspension or mucoadhesive dosage forms.

9. The pharmaceutical composition according to claim 6, further comprising a targeting agent, an agent that improves bioavailability, and/or the agent that controls the delivery.

10. The pharmaceutical composition according to claim 7, further comprising a targeting agent, an agent that improves bioavailability, and/or the agent that controls the delivery.

11. The pharmaceutical composition of claim 8, further comprising a targeting agent, an agent that improves bioavailability, and/or the agent that controls the delivery.

12. The sequence of nucleic acids encoding a peptide compound comprising the amino acid sequence selected from IGKEFKRIVQRIKDFLRNLVPRTE and IGKEFKRIVERIKRFLRELVRPLR.



 

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3 cl, 4 tbl, 6 ex

FIELD: medicine; veterinary.

SUBSTANCE: medicine includes metal iodine and potassium iodide, prolongator and water. 1,2-propylene glycol is used as prolongator, and additionally vitamin A (retinol acetate), vitamin E (alpha-tocopherol acetate), vitamin B1 (thiamine hydrochloride), vitamin B2 (riboflavin), vitamin B6 (pyridoxine hydrochloride), vitamin B12 (cyanocobalamin), iron carbonate, magnium phosphate, manganese sulfate, copper sulfate, zinc sulfate, cobalt chloride, sodium chloride, amber acid, glucose, rectified ethyl alcohol (96%) are applied. Medicine components are taken at the following rate, g/100 ml of distilled water: metal iodine, chemically pure 0.112-0.187; potassium iodide, R 0.337-0.562; vitamin E (alpha-tocopherol acetate) 0.060-0.100; vitamin A (retinol acetate) 3.750-6.250 thousand mass units; vitamin B1 (thiamine hydrochloride) 0.052-0.087; vitamin B2 (riboflavin) 0.037-0.062; vitamin B6 (pyridoxine hydrochloride) 0.034-0.056; vitamin B12 (cyanocobalamin) 0.026-0.044; iron carbonate, R 0.337-0.562; magnium phosphate, R 0.337-0.562; manganese sulfate, R 0.172-0.287; copper sulfate, R 0.090-0.150; zinc sulfate, R 0.315-0.525; cobalt chloride, R 0.071-0.119; sodium chloride, R 0.589-0.981; amber acid, primary standard 0.225-0.375; glucose, AR 0.172-0.287; rectified ethyl alcohol (96%) 0.300-0.500 ml; 1,2- propylene glycol 0.900-1.500 ml. Method involves medicine administration with fodder in dosage of 1.00-1.50 mg per 1 kg of fish weight once per day for 5-7 days.

EFFECT: correction of needs for bioactive substances, prevention of avitaminosis, achievement of high antioxidant organism protection level, prevention of accumulation of non-saturated fatty acid peroxides harmful to fish.

2 cl, 3 tbl, 3 ex

FIELD: medicine; pharmacology.

SUBSTANCE: invention is used for treatment of bacteriemic infections, it is prepared as composition in the form of dry powder, adapted for delution by water with reception of the suspension important pH in a range from approximately 5.0 to approximately 5.5 at initial delution and which in addition contains the stabilizer pH which represents sodium-carboxymethyl cellulose. Besides, the invention concerns application sodium-carboxymethyl cellulose for reduction of degree of degradation clavunalate and for stabilisation pH the received suspension.

EFFECT: stability improvement in preparation.

7 cl, 1 dwg, 1 tbl

FIELD: medicine.

SUBSTANCE: method is implemented as follows: bacterial-enzymatic probiotic "Balance-narine-f" is introduced locally and orally in combination with inhalation of negative air ions.

EFFECT: allows for decrease of complications and higher efficiency of treatment.

1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: sterile aqueous inhalation solution containing active substance Tobramycine. Preparation of invention is offered with high content of active substance (approximately 80 to 120 mg/ml Tobramycine). Preparation also contains acid adjuvant and has low content of sodium chloride (maximum approximately 2 mg/ml). Preparation can be injected or introduced as aerosol with e.g. common sprays.

EFFECT: suitable for application in combination with recent sprays with vibrating membrane and gives the chance for application of an individual therapeutic dose.

24 cl, 1 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: antibacterial and rehydration therapies are combined with prescribed sorbents, symptomatic therapy. From first day of disease prescribed is infusion herb collection consisting of silverweed rhizome, salvia leaves, milfoil herb, St. John's wort herb, inula rhizomes with roots, tickseed herbs, mint leaves, fennel fruits and buckthorn bark at ratio of components 2:2:2:2:2:2:2:2:1. Infusion is taken up dosed 1\3 glass 3 times a day, 30 min before meal within 10 days. Then within three weeks after basic therapy appoint infusion from silverweed rhizome, salvia leaves, milfoil herb.

EFFECT: provides accelerated normalisation of clinical-laboratory indicators, and prevention of infectious complications without by-effects.

2 cl, 1 tbl, 1 ex

FIELD: medicine; biotechnologies.

SUBSTANCE: strain Lactobacillus casei FERM BP-100059 (FERM P-19443), possessing probiotic properties; grow up in the presence of any from one to four amino acids as a source of the nitrogen necessary for growth. At planting in suitable cultural to medium, the final value pH makes 4.0 or lower, and the highest acidity makes 1.5% or above. The strain resistable is to 5% salts of cholic acids. The strain produces an antibiotic. On the basis of the strain as the active beginning, the lactobacillus preparation of probiotic action for humans, animals and plants and the preventive or therapeutic agent against human, animal and plant infections.

EFFECT: ability to colonise and breed in the centres chronic the infections, steady against treatment, strong clearing action and destruction causing a bacterium infection.

12 cl, 5 dwg, 56 tbl, 34 ex

FIELD: medicine; pharmacology.

SUBSTANCE: peroral pharmaceutical dosed out form for treatment of the conditions bound to secretion of acid in a stomach, includes an acid-sensible inhibitor of the proton pump and antagonist of H2-molecular switchers, with the delayed and-or prolonged liberation of the proton pump acid-sensible inhibitor, and fast liberation of antagonist of H2-molecular switchers.

EFFECT: maximum suppression of acid in a stomach after the first dose and throughout all course of treatment.

69 cl, 4 dwg, 7 ex

FIELD: medicine; veterinary science.

SUBSTANCE: vaccine includes inactivated adhesive antigens E.coli K88, E.coli K99, E.coli F-41, E.coli 987P and an adjuvant - aluminum hydrate. As the inactivated adhesive antigens E.coli use inactivated filamentous adhesive antigens E.coli K88 ab, E.coli K88 ad, E.coli K88 ad, E.coli K99, E.coli F-41, E.coli Att-25, E.coli 987P with activity level in reaction of the passive hemagglutination peer as 1:2048-4096; 1:1024-2048; 1:256-512; 1:1024-2056; 1:1024-2056; 1:32-64; 1:256-512, accordingly, at a following parity of components, wt. %: inactivated filamentous adhesive antigens E.coli K88 ab, E.coli K88 ad, E.coli K88 ad, E.coli K99, E.coli F-41, E.coli Att-25, E.coli 987P, taken in mass parities 1:(0.8-1.2:(0.8-1.2:(0.8-1.2:(0.8-1.2:(0.8-1.2:(0.8-1.2, accordingly - 55-65, 5.8-6.2%-s' aluminium hydrate the rest.

EFFECT: rising of safety of livestock of agricultural animals.

2 cl, 1 tbl, 8 ex

FIELD: medicine; pharmacology.

SUBSTANCE: perform processing of an elevated part and roots of the primrose plant (Primula macrocalyx Bge.) with an organic dissolvent - ligroin or hexane with the subsequent extraction with acetone and allocation of a target product using chromatography.

EFFECT: increase of output of the product.

4 ex, 2 dwg

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention pertains to genetic engineering, more specifically to chimeric polypeptides, containing an antagonist of growth hormone receptor. The invention can be used in medicine. The binding domain of the growth hormone is modified by substituting glycine amino acid residue in position 120 and is further modified in site 1, where at least one amino acid residue is substituted, which increases affinity of the growth hormone to its binding domain on the growth hormone receptor. The amino acid residue is then conjugated with the ligand-binding domain of the growth hormone receptor, through a peptide linker.

EFFECT: obtaining a highly effective antagonist of the growth hormone receptor with longer half-life, reduced immunogenesity and nontoxicity, compared to known mutant forms.

35 cl, 16 dwg, 1 tbl

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