Antimicrobial peptides containing arginine- and lysine-containing motive
FIELD: medicine, pharmaceutics.
SUBSTANCE: present invention refers to application of peptide of formula (I): ((X)1(Y)m)n where peptide contains 3 to 200 amino acids and where 1, m and n represent integers within 0 to 10; X and Y which can be identical or different, represent cationic amino acids chosen from arginine and lysine in preparing drugs for treating fungal infection.
EFFECT: ensured applicability of peptide for preparing a drug for treating fungal infection.
26 cl, 53 dwg, 2 tbl, 19 ex
In the present invention proposed antimicrobial peptides. In addition, the invention relates to pharmaceutical compositions containing these antimicrobial peptides and to the use of these peptides for the treatment of, inter alia, microbial infections.
One large family of endogenous antimicrobial peptides, beta-defensin, secreted by epithelial cells that line the digestive tract, respiratory and urogenital way higher mammals. They also produced by keratinocytes in the skin. Their primary role is in providing the necessary first line of defense against infection through these paths pathogenic organisms.
Defensin are one of the most studied classes of antimicrobial peptides. This class consists of a cysteine-rich molecules with three disulfide bridges. They are found in plants, insects and a variety of mammals. People have discovered two classes of defensins, which differ from each other in terms of spatial distribution and relationships between the six cysteine residues. The first of the three classes are alpha defensin (six types), which were isolated from neutrophils (HNP1-4, human neutrophil peptide) and in cells of the Panet of the gastrointestinal tract (alpha defensin 5 and 6). The second class, beta-defensin are longer is, more basic and expressed in all mucous membranes in epithelial cells and keratinocytes that line, and/or contain and/or are present in the digestive tract, respiratory and urogenital tracts and the skin. hBD1 (human beta-defensin 1) is secreted constitutively, and human beta-defensin 2, 3 and 4 (hBD2, hBD3 and hBD4) are produced in response to infection or inflammation. Expression and secretion of hBD2 is triggered by bacterial stimulation, in particular, flagellate bacteria (Harder et al, Nature 1997; 387: 861) and IL1 [alpha] and IL1 [beta] (interleukin 1) (Liu et al. J Invest Dermatol. 2002; 118; 275-281). In some areas of tissue the tumor necrosis factor alpha (TNF-alpha) and lipopolysaccharide (LPS) may also play a role in the induction of the expression of hBD2. In vitro experiments revealed that hBD2 active against gram-negative bacteria, such as Escherichia coli (E. coli), and to a lesser extent against gram-positive bacteria, such as Streptococcus pneumoniae (Str. pneumoniae). Also demonstrated biocidal activity of hBD2 in vitro against the yeast Candida albicans. Expression and secretion of hBD3 is induced by bacterial stimulation, TNF-alpha and especially interferon-gamma (IFNγ), which also has a common property of the molecules involved in inflammatory processes.
In addition to the effective constitutive and regulated natural antimicrobial protection of broad-spectrum action is imposed which provide beta-defensin, these molecules, in particular hBD2, also have the ability to mobilize adaptive branches of the immune response by chemotactic effects on immature dendritic cells and T-cell memory (Yang et al. Science. 1999; 286: 525-528).
Importantly, it was found that beta-defensin not only provide protection against infections caused by pathogenic microbes, but also are key in the regulation and maintenance of optimal density and diversity are essential for the body commensal microbial ecosystems, for example, on the skin and in the gastrointestinal tract and genital tract (Ganz. So Nat Rev Immunol. 2003 3(9): 710-20).
The mechanism of action of beta-defensins is that they are mostly non-toxic to host cells at active concentrations. Therefore, beta-defensin involved as potential targets for therapeutics for a wide range of infections. However, the natural forms of defensins technically difficult to obtain in recombinant systems, resulting in low outputs. In addition, there is a growing amount of data to suggest that due to its chemotactic action of beta-defensin are strong inflammatory compounds (Yang et al. Science. 1999; 286: 525-528; Van Wetering et al. Inflamm Res. 2002; 51(1): 8-15; Niyonsaba et al. Curr Drug Targets Inflamm Allergy. 2003; 2(3): 224-231). Taken the together, these factors make natural defensive unsuitable for therapeutic applications.
Beta defensin also highly sensitive to salt (Porter et al. Infect. Immun. 1997; 65(6): 2396-401; Bals et al. J Clin Invest. 1998; 102(5); 874-80; Valore et al. J Clin Invest. 1998; 101(8): 1633-42; Goldmann et al. Cell. 1997; 88(4): 553-609; Singh et al. Proc Natl Acad Sci USA 95(25): 14961-6). For this reason, beta-defensin cannot provide antimicrobial protection for conditions such as cystic fibrosis, where, although respiratory epithelium abundantly produces beta-defensin in response to chronic bacterial infection associated with this condition, they are inactive due to imbalance in ion transport across membranes of the respiratory epithelium, which leads to increased resorbtive cations (in particular, Na+) and increased secretion of chloride (Donaldson SH and Boucher RC Curr. Opin. Pulm. Med. 2003, Nov; 9(6): 486-91; Davies JC Pediatr Pulmonol Suppl. 2004; 26: 147-8).
Thus, there is a need for additional agents that can be used to treat microbial infections.
The authors of the present invention identified peptides, which unexpectedly possess a higher antimicrobial activity compared with the natural defensively.
According to the first aspect of the invention proposed a peptide containing from 3 to about 200 D - and/or L-amino acids, which may be the same or different, where these amino acids selected from the group consisting of hydrophobic amino acids and/or cationic amino acids. This paragraph shall ptid may contain from 3 to about 100 D - and/or L-amino acids, for example from 3 to 50 amino acids D - and/or L-amino acids, including from about 4 to about 50 D and/or L-amino acids.
The peptides according to the invention is useful, inter alia, in the treatment or prevention of microbial infections.
The following aspect of the invention proposed a peptide containing amino acids of the formula I:
where l and m represent integers from 0 to 10, for example from 0 to 5; n represents an integer from 0 to 10; X and Y, which may be the same or different, are an amino acid selected from the group consisting of hydrophobic amino acids and/or cationic amino acids.
In a preferred aspect of the invention, the peptide contains from 3 to 200 amino acids, for example 3, 4, 5, 6 or 7 up to 100 amino acids, including 3, 4, 5, 6 or 7 up to 20, 25, 30, 35, 40 or 42 amino acids.
The peptide according to the invention may contain from 100 to 200 amino acids, from 27 to 100 amino acids, from 28 to 86 amino acids, from 7 to 27 amino acids, or from 3 to 14 amino acids.
Preferably, the peptides contain from 3 to 15 amino acids, for example from 3 to 7 amino acids.
In the following preferred aspect, the peptides contain one or more than one cysteine residue, for example up to 6 cysteine residues, such as 1, 2, 3, 4, 5 or 6 cysteine residues.
In a preferred aspect of the invention proposed a peptide containing s is necessaty formula II:
where C represents cysteine, l, n and m represent integers from 0 to 10; and X and Y, which may be the same or different, are an amino acid selected from the group consisting of hydrophobic amino acids and/or cationic amino acids.
In the following preferred aspect of the invention proposed a peptide containing amino acids of formula III:
where, X, Y, l, m and n are such as defined in this specification.
In another preferred aspect of the invention proposed a peptide containing amino acids of the formula IV:
where, X, Y, l, m and n are such as defined in this specification.
Since the peptides according to the invention are simpler in structure than natural beta-defensin, they are simple and efficient to obtain. These peptides also essentially insensitive to salt and are not hepatotoxic. In addition, the mechanism of their action, which is physical rather than metabolic (i.e. immediate rupture of membranes, rather than the impact on the components of vital metabolic pathways), minimizes, if not eliminates, the possibility that microorganisms targets may develop resistance to these antimicrobial agents.
As is well known experts who am in this field, amino acids can be placed in different classes based primarily on the chemical and physical properties of amino acid side chain. For example, some amino acids are generally considered to be hydrophilic or polar amino acids, while others are considered to be hydrophobic or nonpolar amino acids. Used in this description, the terms "hydrophobic" and "cationic" may refer to the amino acids having a hydrophobicity greater than or equal to - 1,10, and/or net charge that is greater than or equal to 0, as described in Fauchere and Pliska Eur. J. Med Chem. 10: 39 (1983). Hydrophobic or nonpolar amino acid can also refer to the amino acid having a side chain that is uncharged at physiological pH, which is non-polar and which usually pushes water solution.
In a preferred aspect of the invention X and/or Y is selected from the group of hydrophobic amino acids consisting of glycine, leucine, phenylalanine, Proline, alanine, tryptophan, valine, isoleucine, methionine, tyrosine and threonine, and/or from the group of cationic amino acids, consisting of ornithine, histidine, arginine and lysine. X and/or Y can be a D - or L-amino acids. In addition, X and/or Y may represent an alternating amino acids.
The invention also includes the known isomers (structural article is REO-, conformational and configurational and structural analogues of the above amino acids, as well as those that are modified either by nature (for example, post-translational modification), or by chemical means, including, but not limited to, phosphorylation, glycosylation, sulfonylurea and/or hydroxylation.
Typically, the peptides according to the invention do not include amino acids: aspartic acid, glutamic acid, asparagine, glutamine or serine, but some of the peptides according to the invention can have activity, even if these amino acids are present.
The peptides according to the invention may include one or more than one additional amino acid residue adjacent to one or both terminal cysteine residues of the formula II, III or IV, for example, the peptides can contain up to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) additional amino acid residues. Preferably, the additional amino acids are nezateylivye residues. More preferably, the additional amino acids represent the X and/or Y.
In addition, the amino acid sequence of the peptide can be modified, resulting in a peptide variant that includes replacing at least one amino acid residue in the peptide other amino acid residue, the key replacement, using D-but not L-form.
One or more than one of the residues of the peptide can be replaced by another to change, enhance or preserve the biological activity of the peptide. Such a variant can have, for example, at least about 10% of the biological activity of the corresponding variant peptide. Often use conservative amino acids, that is, replacement of amino acids with similar chemical and physical properties, as described above.
Therefore, for example, conservative amino acid substitutions may include the substitution of lysine for arginine, ornithine or histidine; the substitution of one hydrophobic amino acid for another. After the introduction of the replacement options are screened for biological activity.
The peptide may contain at least 4 amino acids, for example from 4 to 50 amino acids, or 4 and 50 amino acids, for example from 20 to 45 amino acids, for example 20, 25, 30, 35, 40, 42 or 45 amino acids.
In a preferred aspect of the invention X and Y are the same and represent leucine or glycine.
In the following preferred aspect of the invention X represents leucine, and Y represents glycine.
In a preferred aspect of the invention X and Y are the same and represent a lysine or arginine. Thus, in the proposed invention the peptides selected from floor is-L-lysine, poly-D-lysine, poly-L-arginine and poly-D-arginine.
In another preferred aspect of the invention X represents lysine, and Y is arginine.
The peptide according to the invention l and m can be equal to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and n may be equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
The peptide according to the invention l may be equal to 1, n may be equal to 1, and m can be between 4 and 9, for example, m may be equal to 3, 4, 5, 6, 7, 8 or 9.
The peptide according to the invention l, n and/or m can be between 1 and 5, for example 1, 2, 3, 4 or 5.
Preferably, the peptide is acyclic. The peptide may be straight chain or a linear or branched.
The term "peptide"as used herein, refers in a General sense, many amino acid residues joined together by peptide bonds. It is used interchangeably and mean the same thing polypeptide and a protein.
In one embodiment of the invention, the peptide contains an amino acid sequence selected from the group consisting of:
The following aspect of the invention, the peptide contains at least one of the C amino acid sequences (i)to(viii) and an additional amino acid residues, adjacent to one or both of the terminal cysteine residues. Thus, in the following embodiment of the invention proposed a peptide containing amino acid sequence selected from the amino acid sequence presented in figure 1.
The peptides according to the invention, as a rule, are synthetic peptides. These peptides may represent a selected, purified peptides or their variants, which can be synthesized in vitro, for example, by the method of solid-phase peptide synthesis, peptide synthesis catalyzed by enzymes or by using recombinant DNA technology.
For the identification of active peptides that have a small undesirable toxicity or non-toxic to mammalian cells, can be obtained from individual peptides or peptide libraries, and individual peptides or peptides from these libraries can be subjected to screening for antimicrobial activity and toxicity, including, but not limited to, antifungal, antibacterial, antiviral, Antiprotozoal, antiparasitic activity and toxicity.
The peptides according to the invention can exist in various forms, such as free acids, free bases, esters and other prodrugs, salts and tautomers, for example, and the invention included the CE variant forms of these compounds.
Thus, the invention encompasses a salt or prodrug peptide or peptide variant according to the invention.
The peptides according to the invention can be introduced in the form of pharmaceutically acceptable salts. Pharmaceutically acceptable salts of the present invention can be synthesized from the original peptide, which contains a basic or acidic group, standard chemical methods. Generally, such salts can be obtained by interaction of the form of the free acid or base of these peptides with the stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in mixtures of these two solvents; as a rule, are preferred non-aqueous environment, such as ethyl ether, ethyl acetate, ethanol, isopropanol or acetonitrile. Lists of suitable salts are found in Remington''s Pharmaceutical Sciences, 17thed., Mack Publishing Company, Easton, Pa., US, 1985, p.1418, the description of which is included in this invention by reference; see also Stahl et al, Eds, "Handbook of Pharmaceutical Salts Properties Selection and Use", Verlag Helvetica Chimica Acta and Wiley-VCH, 2002.
In the invention, therefore, incorporated pharmaceutically acceptable salts of the described peptides, where the parent compound is modified by obtaining its acidic or basic salts, for example, conventional non-toxic salts or the Quaternary ammonium salts to the e are formed, for example, from inorganic or organic acids or bases. Examples of such salts accession acids include acetate, adipate, alginate, aspartate, benzoate, bansilalpet, bisulfate, butyrate, citrate, comfort, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulphate, aconsultant, fumarate, glucoheptonate, glycyrrhizinate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonic, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, and toilet undecanoate. Salts of the bases include ammonium salts, alkali metal salts, such as salts of sodium and potassium, salts of alkaline earth metals such as calcium salts and magnesium salts, salts with organic bases, such as salts dicyclohexylamine, N-methyl-D-glutamine, and salts with amino acids such as arginine, lysine, etc. Basic nitrogen-containing groups can also be stereoselectivity of such agents as lower alkylhalogenide, such as methyl-, ethyl-, propyl - and butylchloride, bromides and iodides; diallylsulfide, such as dimethyl-, diethyl-, dibutil and dimycolate; long chain halides such as decyl-, lauryl-, myristyl and sterilgarda, bromide and iodide is, aralkylated, such as benzyl and phenetermine, and others.
Salts of carboxyl groups of the peptide or peptide variant according to the invention can be obtained in the usual way by bringing peptide into contact with one or more equivalents of a desired base such as, for example, the base constituting the metal hydroxide such as sodium hydroxide; a carbonate or bicarbonate of a metal, such as, for example, carbonate or sodium bicarbonate; or an amine base such as, for example, triethylamine, triethanolamine and the like.
N-acyl derivatives of an amino group of the peptide or peptide variants according to the invention can be obtained by using N-acylamino amino acid for the final condensation, or by acylation of protected or unprotected amino acids. O-acyl derivatives can be obtained, for example, by acylation of the free hydroxypatite or peptide resin. Any acylation can be performed using standard alleluya reagents, such as acylhomoserine, anhydrides, illimitably and the like.
In the invention includes prodrugs for the active pharmaceutical species described peptides, for example, in which one or more functional groups are protected or converted, but can be transformed in vivo into the functional group, as in the beam of esters of carboxylic acids, convert in vivo to the free acid or in the case of protected amines, to a free amino group. The term "prodrug", as used herein, is presented in the specific structures that are rapidly transformed in vivo to the original structure, for example, by hydrolysis in blood. A detailed discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol.14 of the ACS Symposium Series, Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987; H. Bundgaard, ed., Design of Prodrugs, Elsevier, 1985; and Judkins, et al. Synthetic Communications, 26 (23), 4351-4367 (1996), where each of the publications included in this description by reference.
Thus, prodrugs include drugs having a functional group which is transformed into its reversible derived. Typically, such prodrug into an active drug by hydrolysis. As examples we can mention the following:
|Functional group||Reversible derived|
|Carboxylic acid||Esters, including, for example, aryloxyalkyl esters, amides|
|Alcohol||Esters, including, for example, sulfates and phosphates and esters is Urbanovich acids|
|Amin||Amides, carbamates, imine, enamine|
|Baronova acid||Ester diol|
|Carbonyl (aldehyde, ketone)||Imine, oximes, acetals/ketals, enol complex esters, oxazolidine and tizoxanide|
Prodrugs also include compounds into the active drug by oxidation or reduction reactions. As examples can be mentioned:
- Oxidative deamination
- Sulfoxide recovery
- Disulfide recovery
- Biobotanical alkylation
As the metabolic activation of prodrugs should also mention nucleotide activation, activation by phosphorylation and activation by decarboxylation.
The use of protective groups is fully described in 'Protective Groups in Organic Chemistry', edited by J W F McOmie, Plenum Press (1973)and 'Protective Groups in Organic Synthesis', 2nd edition, T W Greene & P G M Wutz, Wiley-Interscience (1991).
Thus, professionals in this field should understand that, although saxeseni the derivatives of the described peptides may not possess pharmacological activity as such, they can be administered, for example, parenteral or oral route, after which they are metabolized in the body to form compounds that are pharmacologically active. Such derivatives, thus, are examples of "prodrugs". All prodrugs described compounds included in the scope of the invention.
The following aspect of the invention proposed pharmaceutical composition comprising a pharmaceutically effective amount of at least one of the peptides according to the invention or two or more than two different peptides according to the invention.
These peptides also include a pharmaceutically acceptable carrier, excipient or diluent. The expression "pharmaceutically acceptable" is used in this description relative to compounds, materials, compositions and/or dosage forms which are, within the scope of the judgments of experts in the field of medicine, suitable for use in contact with the tissues of humans or perhaps animals without excessive toxicity, irritation, allergic reactions or other problems or complications, in accordance with a reasonable ratio of benefit/risk.
The peptides according to the invention is useful, inter alia, as an antimicrobial peptides, for example, against bacteria, fungi, yeast, parasites, protozoa and viruses. The term "antimicrobial peptide" can be the to use this description to identify any peptide, which has bactericidal and/or bacteriostatic activity and encompasses, without limitation, any peptide described as possessing antibacterial, antifungal, antinicotine, antiparasitic, Antiprotozoal, antiviral, anti-infectious, antiseptic and/or germicidal, algaecide, amebicide, antimicrobial, bactericidal, fungicidal, parasiticide, protozoacidal properties.
Thus, the invention additionally offered a peptide according to the invention for use as a drug. The peptides according to the invention can be used as antimicrobial agents in vivo and ex vivo.
In a preferred aspect of the invention proposed the use of a peptide according to the invention in the manufacture of a medicine for the treatment of microbial infections.
Under "microbial infection" refers to an infection caused by bacterial, parasitic, simplest, viral or fungal pathogen. "Pathogen" is generally defined as any organism that causes disease.
Bacterial pathogen can occur from a bacterial species selected from the group consisting of: Staphylococcus spp., for example, Staphylococcus aureus, Staphylococcus epidermidis; Enterococcus spp., for example, Enterococcus faecalis; Streptococcus pyogenes; Listeria spp.; Pseudomonas spp.; Mycobacterium spp., for example Mycobacterium tuberculosis; Enterobacterspp.; Campylobacter spp.; Salmonella spp.; Streptococcus spp., for example Streptococcus group a or b, Streptococcus pneumoniae; Helicobacter spp., for example, Helicobacter pylori; Neisseria spp., for example, Neisseria gonorrhea, Neisseria meningitidis; Borrelia burgdorferi; Shigella spp., for example Shigella flexneri; Escherichia coli; Haemophilus spp., for example Haemophilus influenzae; Chlamydia spp., for example, Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci; Francisella tularensis; Bacillus spp., for example Bacillus anthracis; Clostridia spp., for example Clostridium botulinum; Yersinia spp., for example, Yersinia pestis; Treponema spp.; Burkholderia spp.; for example, Burkholderia mallei and pseudomallei.
Viral pathogen may occur from a virus selected from the group consisting of: human immunodeficiency virus (VIC and 2); virus T-cell human leukemia (HTLV 1 and 2); Ebola virus; human papilloma virus (for example, HPV-2, HPV-5, HPV-8, HPV-16, HPV-18, HPV-31, HPV-33, HPV-52, HPV-54, HPV-56); papovavirus; rhinovirus; poliovirus; herpesvirus; adenovirus; Epstein-Barr; influenza virus, hepatitis b and C virus of smallpox, rotavirus or coronavirus SARS (severe acute respiratory syndrome).
Parasitic pathogen may occur from parasitic pathogen selected from the group consisting of Trypanosoma spp. (Trypanosoma cruzi, Trypansosoma brucei), Leishmania spp., Giardia spp., Trichomonas spp., Entamoeba spp., Naegleria spp., Acanthamoeba spp spp., Schistosoma spp., Ptasmodium spp., Crytosporidium spp., Isospora spp., Balantidium spp., Loa Loa, Ascaris lumbricoides, Dirofilaria immitis, Toxoplasma spp., for example, Toxoplasma gondii.
Fungal pathogen may occur from a fungal pathogen of the genus Candida spp (for example, C.albicans), Epidermophyton spp., Exophiala spp., Microsporum spp., Trichophyton spp. (for example, T.rubrum and T.interdigitale), Tinea spp., Aspergillus spp., Blastomyces spp., Blastoschizomyces spp., Coccidioides spp., Cryptococcus spp., Histoplasma spp., Paracoccidiomyces spp., Sporotrix spp., Absidia spp., Cladophialophora spp., Fonsecaea spp., Phialophora spp., Lacazia spp., Arthrographis spp., Acremonium spp., Actinomadura spp., Apophysomyces spp., Emmonsia spp., Basidiobolus spp., Beauveria spp., Chrysosporium spp., Conidiobolus spp., Cunninghamella spp., Fusarium spp., Geotrichum spp., Graphium spp., Leptosphaeria spp., Malassezia spp., Mucor spp., Neotestudina spp., Nocardia spp., Nocardiopsis spp., Paecilomyces spp., Phoma spp., Piedraia spp., Pneumocystis spp., Pseudallescheria spp., Pyrenochaeta spp., Rhizomucor spp., Rhizopus spp., Rhodotorula spp., Saccharomyces spp., Scedosporium spp., Scopulariopsis spp., Sporobolomyces spp., Syncephalastrum spp., Trichoderma spp., Trichosporon spp., Ulocladium spp., Ustilago spp., Verticillium spp., Wangiella spp.
Microbial infection to be treated with the peptides of the present invention, can be selected from any human bacterial, fungal, parasitic, enveloped viral pathogens are presented in Table 1.
Thus, the invention proposed the use of a peptide according to the invention in the manufacture of a medicine for the treatment of microbial infection, where the microbial infection is a systemic, local, subcutaneous, cutaneous fungal infection or fungal infection of the mucous membrane.
Fungal infections can be classified as systemic, which means that the infection is deep and affects the internal organs or tolerated by blood, or local (dermatophyte), who appoints, that the infection is superficial and is observed on the skin. In addition, yeast infections can affect the mucous membranes of the body. Yeast infections can also be systemic (e.g., candidemia and other often fatal disease). Fungal infections on the skin are usually treated with creams or ointments (local antifungal medicines). However, systemic infection, yeast infection or local infection that are not resolved after treatment with creams or ointments, may be necessary to treat systemic antifungal drugs (orally or in/in). These drugs are used, for example, for the treatment of common fungal infections such as tinea (ringworm), which is found on the skin, or candidiasis (yeast infection also known as thrush), which can occur in the throat, vagina or other parts of the body. Systemic antifungal drugs also used to treat other deep fungal infections such as histoplasmosis, blastomycosis, and aspergillosis, which can affect the lungs and other organs. Sometimes they are used for the prevention or treatment of fungal infections in people whose immune systems are weakened, such as patients after bone marrow transplantation or organ and people with HIV With the ID.
Local or dermatophyte fungal infection, but typically do not cause death or serious illness is a common and economically important, since treatment can be costly. Local or superficial fungal infection may include infections of the skin, thin plates, the stratum corneum, nails and hair. Skin infections are infections of the skin and nails of the fingers and toes.
In a preferred aspect of the invention fungal infection is an onychomycosis. Onychomycosis can be caused by fungi of the genus Trichophyton spp., for example, this fungus can be a Trichophyton interdigitale or Trichophyton rubrum, but is not limited to this.
The term "onychomycosis" includes, but is not limited to, the distal lateral subungual, white surface, proximal white subungual, secondary dystrophic, primary degenerative, vnutrisajtovoj, candidly (such as onychomycosis and chronic mucocutaneous disease) types of onychomycosis. It is shown that onychomycosis is a significant risk factor for many serious clinical complications, such as acute bacterial cellulitis of the hands/feet and other secondary bacterial infection, thus, the present invention covers these infections.
The peptides according to the invention are effective the governmental antimicrobial peptides for a wide variety of pathogens. However, the peptides according to the invention may also be useful in the treatment of other conditions, including, but not limited to, cystic fibrosis and other conditions associated with infections of the mucous membranes, such as gastrointestinal, genitourinary, or respiratory infections.
The peptides according to the invention may also be useful in the treatment or prevention of, inter alia, wounds, ulcers and lesions, such as skin wounds, such as cuts or burns, and conditions associated with them.
The term "treatment" refers to the effects of the peptides described in this invention, that is, to assist patients affected (infectious) disease, including improvement of symptoms or slow disease progression.
Used in this description, the term "treatment of wounds can include wound healing and associated with it conditions and therapy that stimulates, promotes or accelerates the healing of tissues and includes scars, burns, psoriasis, acceleration remodeling tissue, for example, after cosmetic surgery and transplantation authority.
Thus, in this aspect of the invention proposed a substrate to which is applied or to which it is attached peptide according to the invention. Preferably, this substrate is suitable for application to wounds or delivery to the designated wounded who I am. Preferably, this substrate allows to transfer the peptides according to the invention of the substrate in the wound bed to achieve their antibiotic effect. The substrate may be a nonwoven material, such as dressings for wounds. This dressing material may consist of a textile material, or it can represent collagenopathy material.
The peptides according to the invention can also find application as a disinfectant/sanitizer. In this context, the peptide or pharmaceutical composition according to the invention can be used either alone or in combination with other antimicrobial agents on the surface to be processed. As used herein, the "surface to be processed may be a substrate, as defined herein, or medical device.
The following aspect of the invention, a method of treating or preventing a microbial infection in a subject, comprising the introduction of a specified subject a therapeutically effective amount of a peptide according to the invention.
In the preferred method according to the invention the microbial infection is a fungal infection. In the method according to the invention, the peptide can be applied to the skin or nails of the specified entity.
The mammalian is affected, birds and other animals can be treated with peptides, compositions or methods described in this invention. Such mammals and birds include humans, dogs, cats and livestock, such as horses, cattle, sheep, goats, chickens and turkeys, and the like. In addition, plants can also be treated with peptides, compositions or methods according to the invention.
In cases when the subject is an animal, the method according to the invention can be used to nagtatanim structures, including, but not limited to, hooves, claws and hooves.
The method according to the invention may comprise, in addition to peptide therapy, therapy that can enhance the penetration of the peptides in the nails. This process can be enhanced by chemical or physical means. Physical therapy, such as flaking nails or grinding the dorsal layer of the nail, can enhance the penetration of the peptides according to the invention. Chemical increased permeability of the nail for the peptides according to the invention can be achieved by the destruction of the physical or chemical bonds inside the keratin of the nail plate. Agents, softening the nails, including, but not limited to, urea and salicylic acid to increase hydration of the nail with decreasing density of the nail and, therefore, can increase its permeability to peptides according to the invention. Soy is inane, containing sulfhydryl groups, will cleave disulfide bonds in the keratin of the nail and can lead to destabilization and increased penetration of drugs. Compounds, including but not limited to, acetylcysteine and derivatives mercaptoethanol can be used in combination with the peptides according to the invention. Other known excipients/adjuvants for the permeability of the nail, which can be used in combination with the peptides of the invention include methylsulfonylmethane, urea, polyethylene glycol, N-(2-mercaptopropionyl)glycine, dimethyl sulfone and 2-n-nonyl-1,3-dioxolane.
The following aspect of the invention, a method for treating a wound in a subject, comprising applying to the wound a therapeutically effective amount of the peptide or substrate according to the invention.
The peptides according to the invention, including their salts, are administered in such a way as to reduce at least one symptom associated with infection, the indication or disease, or reducing the number of antibodies bound to the indication or disease.
To achieve the desired(s) effect(s) of the peptide, its variant, or a combination, you can enter as a single or divided doses, for example at least about 0.01 mg/kg to about 500-750 mg/kg, at least about 0.01 mg/kg to about 300 to 500 mg/is g, at least from about 0.1 mg/kg to about 100-300 mg/kg or at least about 1 mg/kg to about 50-100 mg/kg of body weight or at least about 1 mg/kg to about 20 mg/kg of body weight, although other dosages may provide beneficial results. Enter the amount will vary depending on various factors, including, but not limited to, the selected peptide and its clinical effects, disease, weight, physical condition, health status, age of the mammal, must be achieved by prevention or treatment, and whether the peptide is chemically modified. Such factors can be easily determined by the physician in the study of empirical data, clinical trials and the study of preclinical results in animal models or other test systems that are available in this field.
The introduction of therapeutic agents in accordance with the present invention can be implemented in a single dose, in multiple doses, in a continuous or alternating mode depending on, for example, on the physiological state of the recipient, whether the purpose of the introduction of therapeutic or prophylactic, and other factors known to practitioners in this field. The introduction of the peptides according to the invention may be essentially continuous during the course the e pre-selected period of time, or it can be done as a series of divided doses. Consider both local and systemic injection.
For the preparation of compositions of the peptides synthesized or get any other way, purified as necessary or desirable, and then lyophilizer and stabilize. Then, the peptide can be brought to a suitable concentration and possibly combined with other agents. The absolute mass of this peptide is included in the standard dose, can be widely varied. For example, you can enter from about 0.01 to about 2 g or from about 0.01 to about 500 mg of at least one peptide according to the invention or a variety of peptides that are specific for a particular cell type. Alternatively, the standard dosage can vary from about 0.01 g to about 50 g, from about 0.01 g to about 35 g, from about 0.01 g to about 25 g, from about 0.5 g to about 12 g, from about 0.5 g to about 8 g, about 0.5 g to about 4 g, or from about 0.5 g to about 2 g
Daily doses of the peptides according to the invention can also vary. Such doses can be in the range of, for example, from about 0.001 g/day to about 100 or 50 g/day, from about 0.1 g/day to about 25 g/day, from about 0.1 g/day to about 12 g/day, from about 0.1 g/day to about 5 g/day, from about 0.1 g/day to about 2.5 g/day, elprimero 0.1 g/day to about 2 g/day, from about 0.5 g/day to about 8 g/day, from about 0.5 g/day to about 4 g/day, from about 0.5 g/day to about 2 g/day and from about 0.5 g/day to about 1 g/day.
Thus, one or more than one suitable standard dosage form, containing therapeutic peptides according to the invention, can be entered in many ways, including oral, parenteral (including subcutaneous, intravenous, intramuscular and intraperitoneal), rectal, dermal, transdermal, intrathoracic, intra-lungs and intranasal (respiratory) routes. Therapeutic peptides can be prepared in the form of liquid preparation or for sustained release (for example, using microencapsulation, see WO 94/07529 and U.S. patent No. 4962091). Drugs can be, if appropriate, conveniently presented in discrete standard dosage forms, and can be prepared by any of the methods well known in the pharmaceutical field. Such methods may include the stage of mixing therapeutic agent with liquid carriers, solid matrices, semi-solid carriers, finely powdered solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.
When therapeutic peptides according to the invention prigotovlyu the t for oral administration, they are usually combined with pharmaceutically acceptable carrier, diluent or excipient with the formation of a pharmaceutical preparation or a standard dosage forms. For oral administration, the peptides can be presented in the form of powder, granular drug, solution, suspension, emulsion or in a natural or synthetic polymer or resin for ingestion of the active ingredients from a chewing gum. Active peptides can be presented in the form of a bolus, electuary or paste. Oral insertion of therapeutic peptides according to the invention can also be prepared in the form of drugs for sustained release, for example, these peptides can be coated, subjected to microencapsulation or otherwise placed in the device for prolonged delivery. The total active ingredients in these preparations comprise from 0.1 to 99.9 wt.%/wt. drug.
Pharmaceutical preparations containing therapeutic peptides according to the invention can be prepared using methods known in this field, using well known and readily available ingredients. For example, the peptide can be prepared in the form of the drug with the usual excipients, diluents or carriers in the form of tablets, capsules, solutions, suspensions, powders, is erotola or the like. Examples of excipients, diluents, and carriers that are suitable for such drugs include buffers, as well as fillers and diluents, such as starch, cellulose, sugars, mannitol, and derivatives of silicon. May also include binders, such as carboxymethylcellulose, hydroxyethylcellulose, hypromellose and other cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone. Can be enabled moisturizing agents such as glycerin, leavening agents, such as calcium carbonate and sodium bicarbonate. May also include agents, retarding dissolution such as vaseline. May also include resorption accelerators such as Quaternary ammonium compounds. Can be included surface-active agents such as cetyl alcohol and glycerol monostearate. Can be added to the adsorption agents, such as kaolin and bentonite. May also include lubricating agents such as talc, calcium stearate and magnesium and solid polyethylene glycols. Can also be added preservatives. The composition of the invention may also contain thickeners, such as cellulose and/or cellulose derivatives. They may also contain resins, such as xanthan, guar or carbazole or Arabian gum, or, in the alternative, the polyethylene glycols, Antony and montmorillonite and the like.
For example, tablets or caplet containing peptides according to the invention can include buffering agents such as calcium carbonate, magnesium oxide and magnesium carbonate. Suitable buffering agents can also include acetic acid salts, citric acid salts, boric acid salts and phosphoric acid salts. Caplet and tablets can also include inactive ingredients such as cellulose, pregelatinized starch, silicon dioxide, hypromellose, magnesium stearate, microcrystalline cellulose, starch, talc, titanium dioxide, benzoic acid, citric acid, corn starch, mineral oil, polypropylenglycol, sodium phosphate, zinc stearate, and the like. Hard or soft gelatin capsules containing at least one peptide according to the invention can contain inactive ingredients such as gelatin, microcrystalline cellulose, sodium lauryl sulfate, starch, talc, titanium dioxide and the like, as well as liquid solvents, such as glycols (pegs) and vegetable oil. In addition, caplet or tablets covered intersolubility coating containing one or more peptides according to the invention are designed to resist disintegration in the stomach and dissolve in the more neutral to alkaline environment of the duodenum to the women really.
Therapeutic peptides according to the invention can also be prepared in the form of elixirs or solutions for convenient oral administration or as solutions appropriate for parenteral administration, for example, intramuscular, subcutaneous, intraperitoneal or intravenous routes. Pharmaceutical drugs are therapeutic peptides according to the invention can also take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion, or suspension or ointment.
Thus, therapeutic peptides can be prepared in the form of preparations for parenteral administration (e.g. by injection, for example bolus injection or continuous infusion) and may be presented in the form of a standard dose in ampoules, pre-filled syringes, infusion containers of small volume or mnogochasovykh containers. As indicated above, may be added as preservatives to maintain the shelf life of the dosage form. Active peptides and other ingredients may form suspensions, solutions or emulsions in oily or aqueous media and may contain such agents for the preparation of drugs, as suspendida, stabilizing and/or dispersing agents. Alternatively, active peptides and other ingredients may be in powder form, poluchennaya aseptic selection of sterile solid or by lyophilization from solution, for reconstitution with a suitable solvent, for example, sterile pyrogen-free water before use.
These preparations may contain pharmaceutically acceptable carriers, solvents and adjuvants which are well known in the field. It is possible, for example, to prepare solutions using one or more than one organic solvent which is acceptable from the physiological standpoint, chosen, in addition to water, from solvents such as acetone, acetic acid, ethanol, isopropyl alcohol, dimethyl sulfoxide, ethylene glycol ethers such as the products sold under the name "Dowanol", polyglycols and polyethylene glycols, With1-C4alkalemia esters of short chain acids, ethyl - or isopropylacetate, triglycerides of fatty acids, such as the products sold under the name "Miglyol", isopropylmyristate, animal, mineral and vegetable oils and polysiloxane.
Preferably, the pharmaceutical preparations of therapeutic peptides according to the invention can also take the form of a solvent or diluent containing peptide. Solvents or diluents may include acid, dimethyl sulfone, N-(2-mercaptopropionyl)glycine, 2-n-nonyl-1,3-dioxolane and ethyl alcohol. Preferably, the solvent/diluent is an acidic solvent such as acetic acid, citric acid, boric KIS the GTC, lactic acid, propionic acid, phosphoric acid, benzoic acid, butyric acid, malic acid, malonic acid, oxalic acid, succinic acid or tartaric acid.
More preferably, the solvent is a solution of acetic acid. Solvent, for example a solution of acetic acid, may be present in the composition at a concentration of less 1%, 0,5%, 0,25%, 0,1%, 0,05% or 0.01% of acid, for example acetic acid.
The following aspect of the present invention proposed the use of acid in the manufacture of a medicine for the treatment of microbial infections, particularly fungal infections. This fungal infection can be a onychomycosis. Onychomycosis can be caused by fungi of the genus Trichophyton spp., but not limited to, such as this fungus can be a Trichophyton interdigitale or Trichophyton rubrum. Acid may be the same as described in this invention above. Preferably, the acid is an acetic acid. More preferably, the acid is present in solution at a concentration of less 1%, 0,5%, 0,25%, 0,1%, 0,05% or 0.01% of acid, for example acetic acid. Typically this drug adapted for local injection for the treatment of, for example, nails.
As used in this specification, hereinafter, the term "active agent" includes a single PE is Ted according to the invention or combination of peptides, as described in this invention. The term "active agent" may also encompass pharmaceutically effective amount of acid, as described in this invention. Active agents can be administered simultaneously, sequentially or separately. In General, it is preferable that such introduction was local.
Active agents can be introduced in synergistically effective amounts. Thus, the invention includes: applying a synergistically effective amounts of active agents, such as peptide according to the invention and a pharmaceutically effective amount of acid, as described in this invention, for the manufacture of a product such as a medicinal product for simultaneous, separate or sequential introduction of these agents in the treatment of microbial infection.
It is possible to add, if necessary, adjuvant chosen from antioxidants, surfactants, other preservatives, film-forming, keratolytic or comedolytic agents, flavorings, corrigentov and dyes. Can be added antioxidants such as tert-butylhydroquinone, bottled hydroxyanisol, bottled hydroxytoluene and α-tocopherol and derivatives thereof.
Also considered combination products that include one or more of the peptides of the present invention and one or more friend is x antimicrobial or antifungal agents, for example, a polyene, such as amphotericin b lipid complex amphotericin b (ABCD), liposomal amphotericin b (L-AMB) and liposomal nystatin, azoles and triazoles, such as voriconazole, fluconazole, ketoconazole, Itraconazole, Posaconazole, and the like; inhibitors glucosidase, such as caspofungin, micafungin (FK463) and V-echinocandin (LY303366); grizeofulvina; allylamines, such as terbinafine; flucytosine or other antifungal agents, including those described in this invention. In addition, provide that the peptides can be combined with local antifungal agents such as ciclopirox alamin, haloprogin, tolnaftate, undecylenic, local nystatin, amorolfine, butenafine, naftifine, terbinafine and other local agents.
In addition, peptides are well suited for the manufacture of dosage forms with prolonged release, and the like. These drugs can have such a composition that they release the active peptide, for example, in the specific area of the intestinal tract or the respiratory tract, possibly over a period of time. Coatings, envelopes, and protective matrices may be made, for example, polymeric substances, such as polylactic glycolate, liposomes, micro-emulsions, microparticles, nanoparticles, or waxes. These coatings, envelopes and protective matrices are useful for coating p is permanent devices for example, stents, catheters, tubing for peritoneal dialysis, drainage devices, and the like.
For the local introduction of active agents can be prepared in the form of drugs, as is well known in this field, for direct application to the target area. Forms, mainly adapted for local use, have the form of, for example, creams, milk, gels, powders, dispersions or microemulsions, lotions, thickened to a greater or lesser extent, impregnated pads, ointments or sticks, aerosol products (e.g., sprays or foams), Soaps, detergents, lotions, or pieces of soap. Other conventional forms for this purpose include dressings for wounds, covered with bandages or other polymeric coatings, ointments, creams, lotions, pastes, jellies, sprays and aerosols. Thus, therapeutic peptides of the invention can be delivered via patches or bandages for skin injection. Alternatively, the peptide can be prepared in the form of the drug, which is a part of an adhesive polymer, such as polyacrylate or a copolymer of acrylate/vinyl acetate. For long-term applications it may be desirable to use a micro-porous and/or permeable underlying layered materials, in order to minimize hydration or maceration of the skin. On strause layer can have any suitable thickness, which will provide the desired protective and supportive functions. A suitable thickness will be typically from about 10 to about 200 microns.
Local injection can be done in the form of a coating or nail Polish. For example, the antifungal peptides can be prepared in the form of a solution for local injection, which contains ethyl acetate (NF (national formulary), isopropyl alcohol (USP, USP) and butyl monoether poly[metilidinovy ether/maleic acid] in isopropyl alcohol.
Pharmaceutical agents for local injection can contain, for example, a physiologically acceptable buffered saline solution containing from about 0.001 mg/ml to about 100 mg/ml, for example from 0.1 mg/ml to 10 mg/ml of one or more peptides of the present invention specific for the indication or disease to be treated.
Ointments and creams can be prepared, for example, in the form of drugs with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions can be prepared in the form of medication with water or oil based, and they will generally also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspendida agents, thickening agents, or coloring agents. Active the peptides can also be delivered via iontophoresis, for example, as disclosed in U.S. patent No. 4140122; 4383529 or 4051842. The percentage weight of therapeutic agent according to the invention, present in the local drug will depend on various factors, but generally will be from 0.01% to 95% of the total mass of the drug and typically about 0.1-85% by mass.
Drops, such as eye drops or nose drops, may be prepared in the form of drugs with one or more therapeutic peptides in aqueous or nonaqueous basis, also containing one or more dispersing agents, solubilizing agents or suspendida agents. Liquid sprays can be introduced by a pump or convenient to be delivered from the package under pressure. Drops can be delivered using a simple bottle, closed drip, plastic bottles, adapted to drip delivery of liquid content, or by using the cap of a special form.
In addition, therapeutic peptide can be prepared in the form of a preparation for local injection in the mouth or throat. For example, drugs active ingredients can be prepared in the form of cakes, optionally containing a corrective basis, usually sucrose and Arabian gum or tragakant; pastilles containing composition in an inert basis such as gelatin and glycerin or sucrose and Arabian gum; and gargle for mouth containing composition is Yu of the present invention in a suitable liquid carrier.
The pharmaceutical preparations of the present invention may include as a possible ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that is available in this area. Examples of such substances include normal saline solutions, such as physiologically buffered salt solutions and water. Specific non-limiting examples of carriers and/or diluents that are useful in the pharmaceutical preparations of the present invention include water and physiologically acceptable buffered saline solutions such as phosphate-saline buffer solution, pH 7.0 to 8.0.
The peptides according to the invention can also enter the respiratory tract. Thus, the present invention also suggested that aerosol pharmaceutical preparations and dosage forms for use in the methods according to the invention. Typically, such dosage forms contain a quantity of at least one of the agents according to the invention, effective to treat or prevent the clinical symptoms of a specific infection, indications or disease. Any statistically significant attenuation of one or more symptoms of the infection, indications or disease, which was treated according to the method of the present invention, is considered as the treatment that the first infection, indications or disease within the scope of the invention.
Alternatively, for administration by inhalation or insufflation, the composition may take the form of a dry powder, for example a powder mix of a therapeutic agent and a suitable powder base, such as lactose or starch. The powder composition may be presented in a standard dosage form, for example, capsules or cartridges, or, for example, gelatin or blister packs from which the powder can be entered using the inhaler, insufflator or metered dose inhaler (see, for example, metered-dose inhaler under pressure (MDI) and dry powder inhaler, unveiled in Newinan, S.P. in Aerosols and the Lung, Clarke, S.W. and Davia, D. eds., p.197-224, Butterworths, London, England, 1984).
therapeutic peptides of the present invention can also enter in an aqueous solution with the introduction in the form of an aerosol or inhaled form. Thus, other aerosol pharmaceutical preparations may contain, for example, a physiologically acceptable buffered saline solution containing from about 0.001 mg/ml to about 100 mg/ml of one or more peptides of the present invention specific for the indication or disease to be treated. Dry aerosol in the form of finely dispersed solid particles, peptide or nucleic acids which do not dissolve the sludge is not suspendered in the liquid, also useful in the practice of the present invention. Preparations of peptides of the present invention can be prepared in the form of powders, and can contain finely ground particles having an average particle size of from about 1 to 5 microns, alternatively from 2 to 3 μm. Finely ground particles can be obtained by comminution and filtering through a sieve, using techniques well known in the field. These particles can be administered by inhalation of a given quantity of finely powdered substance, which may be in powder form. It should be understood that the standard content of the active ingredient or ingredients contained in an individual dose of each aerosol dosage forms, does not necessarily constitute an effective amount for treating a particular infection, indications or disease, since the necessary effective amount can be achieved by introducing a variety of standard doses. In addition, the effective amount can be achieved using lower doses than in a conventional dosage form, either individually or in a series of introductions.
With regard to the introduction in the upper (nasal) or lower respiratory tract by inhalation, therapeutic peptides according to the invention is conveniently delivered from a nebulizer or packing under pressure is m, or other convenient means of delivering an aerosol spray. Packing under pressure may contain suitable propellant, such as DICHLORODIFLUOROMETHANE, Trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of an aerosol under pressure standard dose can be determined by providing a valve to deliver a measured quantity. The nebulizers include, but are not limited to, nebulizers, described in U.S. patent No. 4624251; 3703173; 3561444 and 4635627. Aerosol delivery systems of this type, as disclosed in this invention are available from various commercial sources, including Fisons Corporation (Bedford, Mass.), Schering Corp. (Kenilworth, NJ) and American Pharmoseal Co. (Valencia, CA). As for intranasal administration, therapeutic agent can also enter via nose drops, a liquid spray, for example, by means of a spray, representing a plastic bottle, or metered dose inhaler. Types of spray guns are Mistometer (Wintrop) and Medihaler (Riker).
In addition, the active ingredients can also be used in combination with other therapeutic agents, such as pain relievers, anti-inflammatory agents, antihistamine agents, bronchodilators, and the like, either in this state or any other state.
Candidate peptides can be obtained from libraries of peptides according to the invention, as described in this invention. The peptides can also be individually or rationally constructed as having a specific structural feature.
Below the invention will be described only with the help of example with reference to the following figures:
figure 1 shows amino acid sequences of four peptides according to the invention;
figure 2 presents a histogram showing the growth of the fungus T. interdigitale through (a) 4 days and (b) 7 days after treatment with the peptide of Figure 1;
figure 3 presents a histogram showing the growth of the fungus T. rubrum through (a) 4 days and (b) 7 days after treatment with the peptide of Figure 1;
figure 4 presents a histogram showing the growth of the fungus Candida albicans through (a) 24 hours and (b) 48 hours after treatment with the peptide of Figure 1;
figure 5 presents a histogram showing dose-dependent experiment for peptide 1 (shown in figure 1) on the survival of Candida albicans after 24 hours on the Le processing;
figure 6 presents a graph showing the survival of Candida spp. after 24 hours in the presence of a number of doses of peptide 4, as shown in figure 1;
figure 7 presents a graph showing the survival rate of 3 different strains of bacteria after 24 hours in the presence of a number of doses of peptide 4, as shown in figure 1;
on Fig presents a histogram showing the synergistic effect of 0.01% acetic acid for antifungal activity against T. rubrum) peptide 4 (1 mg/ml) on day 3 of growth;
figure 9 presents a histogram showing inhibition of T. interdigitale and T. rubrum peptide 4;
figure 10 presents a histogram showing the effects of peptide 3 and 4 on T. interdigitale;
figure 11 presents a histogram showing the effect of acetic acid on the growth of T. interdigitale;
on Fig presents a histogram showing the effect of polylysine on the growth of T. interdigitale;
on Fig presents a histogram showing the effects of polylysine and polyalanine on the growth of T. rubrum;
on Fig presents a histogram showing inhibition of T. interdigitale and T. rubrum poly-L-arginine;
on Fig presents a histogram showing the effect of low concentrations of polyalanine on T. rubrum and T. interdigitale;
on Fig presents a histogram showing the effect of trimers on the growth of T. rubrum;
on Fig presents the histogram, the demo is stereoma effect of peptide 4 and NaCl on the growth of T. Interdigitale;
on Fig presents a graph showing the effect of peptide 4 on Candida albicans at high concentrations of salt;
on Fig presents a histogram showing the effects of polylysine and polyalanine on the survival of Candida albicans.
Table 1 lists the human bacterial, fungal, parasitic and viral envelope pathogens that are subject to treatment with the peptides according to the invention.
In Table 2 are described in detail peptides, which correspond to the amino acid polymer codes shown in the Results and Figures.
Below the invention will be described by reference only to the following examples.
Materials and methods reagents
All peptides were obtained either by solid-phase synthesis under contract with Invitrogen-Evoquest, Carlsbad, CA, USA, or received from the supplier peptides NeoMPS SA (Strasbourg, France) or Sigma-Aldrich Chemical Company Ltd. (Poole, UK). As for the tests with fungi, the lyophilized peptide is prepared in the form of an initial solution of 1000 µg/ml in buffer for analysis. In cases where it is clearly shown in experiments on the basis of which received 2 to 8 and 11, acetic acid was added as a solvent to a final concentration of 0.5%.
Strains of Trichophyton interdigitale (NCPF 117) and Trichophyton rubrum (NCPF 335) were obtained from National collection of pathogenic fungi in Bristol and was maintained at ku is ture by reseeding with approximately monthly intervals on the sloped agar Saburo (Sabuoraud) and potato dextrose agar at 30°C. Candida albicans strain 3179 (obtained from National collection of type culture [NCTC], Colindale) supported in broth Mueller-Hinton (Mueller Hinton firms Oxoid at 37°C. Streptococcus pyogenes strain 8198, Staphylococcus aureus strain 10642 (methicillin-resistant) and E. coli 0157 strain 12900 were obtained from NCTC in Colindale and maintained in broth Mueller-Hinton firm Oxoid at 37°C.
Sensitivity analyses fungus growth
To determine the sensitivity of fungal strains for each of the tested peptides, their impact on the growth of fungi was evaluated as follows. Suspensions of conidia and hyphal fragments T. interdigitale and T. rubrum were prepared by adding 10 ml of fresh nutrient glucose broth (Nutrient Glucose Broth, NGB) (nutrient broth Oxoid company, containing 2 wt.%/about. glucose) culture on the sloped agar and mixed with a spatula. The resulting suspension fragments of spores/hyphae was filtered through 2 layers of sterile surgical gauze to remove large films hyphae and pieces of agar. 20 μl of this suspension (absorption at 540 nm approximately 0.1, which corresponds to about 106germs/ml) was inoculable in each well of a sterile 96-well microtiter tablets, which beforehand was added to a total volume of 80 µl of culture medium (NGB) and the corresponding quantity of a solution of the peptide. Control wells consisted of wells, which are a finite amount of buffer for analysis, 100 μl drove one environment NGB plus solvent (if it was used, and in the same concentration as in the sample peptides). Monitoring the growth of fungi in the tablets was carried out on the basis of the absorption at 540 nm in the reader for tablets Microtek after 24 h, 4 days and 7 days incubation at 30°C.
Tests on the survival of Candida albicans
To determine the sensitivity of strains of fungi for each of the tested peptides, their impact on the survival of Candida were evaluated as follows. Culture of C. albicans were grown for 18-24 hours, then kept at 4°C until use. Fresh night culture was centrifuged at 2000×g for 10 min and washed with fresh broth Mueller-Hinton, bringing the number of viable cells to values between 5×106and 1×107/ml. Buffer for analysis were prepared by adding 100 ál of medium NGB to 6.9 ml of 10 mm sodium phosphate buffer, pH of 7.7. 35 μl of buffer for analysis without peptide or with a range of concentrations of peptides were added in a sterile polypropylene vial with a screw cap was added 15 μl inoculum of Candida albicans, described above. The vials were incubated at 37°C in a water bath for 2 h and the number of survivors of Candida spp. was determined by serial dilution in sterile phosphate-buffered saline (FSB) and were sown in 9 cm Petri dishes containing agar Saburo Phi is we Oxoid (20 ml). Counting was performed after incubation of these cups at 37°C for 18-24 hours
Analyses of survival bacteria
Streptococcus pyogenes strain 8198, Staphylococcus aureus strain 10642 (methicillin-resistant) and E. coli 0157 strain 12900 (all derived from NCTC, Colindale) were grown for 18-24 hours, then kept at 4°C until use. Fresh night culture was centrifuged at 2000×g for 10 min and washed with fresh broth Mueller Hinton. Sensitivity to each of the four peptides was analyzed as described above for C. albicans. For E. coli and S. aureus original number of cells for sensitivity analysis peptide was 108/ml, and the medium used for counting, was a nutrient agar (Oxoid). Str. pyogenes grew worse than other strains on agar Mueller-Hinton, and therefore the original number of cells for these assays was lower than for the other strains, with 106/ml. Survival Str. pyogenes was determined using tryptone-soy agar Oxoid instead of nutrient agar.
Inhibition of growth Trichophtyon spp. peptides 1-4
Two clinically relevant skin pathogenic fungus Trichopyton rubrum and Trichopyton interdigitale were cultured as described in Materials and Methods, only in the growth medium (control culture) or in the growth medium containing 50 μg/ml of peptide 1, 2, 3 or 4 (shown in Fig.1). The growth of T. interdigitale and T. rubrum were estimated p is the measurement of optical density (absorbance at 540 nm) after 4 and 7 days in culture. Compared with the control untreated samples each tested peptide significantly inhibited growth of T. interdigitale (Figure 2) and T. rubrum (Figure 3) at 4 and 7 days. The control culture of each test strain continued to grow, as indicated by the increase of the OD readings between 4 and 7 days.
Inhibition of growth and survival of Candida spp. peptides 1-4
The yeast Candida albicans was cultured as described in Materials and Methods, only in the growth medium (control culture) or in the growth medium containing 50 μg/ml or 100 μg/ml and 300 μg/ml or 500 μg/ml of peptide 1, 2, 3 or 4. The growth of C. albicans was evaluated on the basis of the measurement of optical density (absorbance at 540 nm) after 24 h (Figa) and 48 h (Figb) in culture. Compared with the control untreated samples each tested peptide significantly inhibited growth of C. albicans time - and dose-dependent manner. The dependence of the inhibition of the growth of the dose was further confirmed in experiments in which the growth of C. albicans was assessed optically after 24 h in culture in control conditions (only growth medium) or in the presence of several concentrations of peptide 1, 50 µg/ml to 500 μg/ml (Figure 5). In a separate experiment, the survival of C. albicans was assessed after 18-24 h in cultures grown only in medium (control) or containing a range of concentrations of peptide 4 with an interval of 1 μg/ml to 1000 mcgml (6). Survival of organisms C. albicans, as assessed on the basis of estimates of survival after 24 h in culture, was decreased dose-dependent manner (6).
Inhibition of survival of bacteria peptide 4
Three clinically relevant pathogenic bacteria, E. coli 0157, methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pyogenes were exposed to the effect as described above under Materials and Methods, series, containing a range of concentrations of peptide 4. After 3 h, the samples of each bacterial culture was transferred to plates with a suitable solid-phase growth environment and the number of viable colonies in the control (only growth medium) and treated (growth medium containing the peptide 4) samples were evaluated after 18-24 h After 3 h exposure to peptide 4 significantly inhibited the survival of each bacterial strain (Fig.7) compared to control untreated cultures in a dose-dependent manner.
Acetic acid enhances the antifungal activity of the peptide 4
As the control (no peptide)and test (containing peptide 1, 2, 3 or 4) in the experiments shown in Figure 2 and 3 contained 0.5% of acetic acid as solvent peptide (see Materials and Methods), was assigned a separate experiment, to determine whether itself acetic acid play a role in the activity of PEP the IDA and/or survival of fungi. For this purpose we have done experiments on the growth of T. rubrum, as in section Materials and Methods, only with the growth medium, the growth medium containing only 0.01% of acetic acid, with growth medium containing 1 mg/ml peptide 4, and with growth medium containing 1 mg/ml peptide plus 4 0,01% acetic acid. The growth of the fungus was determined by OD as described above, after 3 days in culture. As expected, peptide 4 inhibited the growth of T. rubrum. Only 0.01% of acetic acid had no significant effect on growth of T. rubrum (Fig), but when included in an environment with peptide 4 the presence of 0.01% acetic acid significantly inhibited growth of T. rubrum more than 1 mg/ml of a single peptide 4.
Inhibition of growth of T. interdigitale and T. rubrum peptide 4
Inhibitory effects of peptide 4 on the growth of Trichophyton spp. was determined by analyzing the growth of fungus, as in Materials and Methods. T. rubrum and T. interdigitale was cultured in the same medium or in medium containing 3 different concentrations of peptide 4. Acetic acid was not present in any of the samples. Controls with the same environment used to illustrate the background absorption of the medium. The growth of the fungus was determined by OD as described above, after 96 hours incubation at 30°C. As shown in Fig.9, these analyses confirmed the inhibitory effect of peptide 4 on the growth of both species of fungi, and T. interdigitale constantly more sensitive to inhibitory the effects of treatment with peptide 4, than T. rubrum. The growth of T. interdigitale inhibited at concentrations of peptide 0.55 mg/ml
Effects of peptides 3 and 4 on the growth of T. interdigitale
Evaluated the antifungal potential of peptide 3 and peptide 4 in respect of T. interdigitale. Analyses of growth inhibition were performed as in Materials and Methods, in the absence of acetic acid. Since the peptides 1-3 are vysokoshirotnymi and, therefore, insoluble, previously only tested against Trichophyton spp. in acetic acid as solvent. When the culture of T. interdigitale were grown for 7 days in the presence of peptide 3, peptide 4, or one environment without solvent of acetic acid and measured the increase in OD was observed that peptide 4 significantly inhibited growth of the fungus (Figure 10), while peptide 3 did not show inhibitory activity (Figure 10). This increased activity of cationic peptide 4 in comparison with the hydrophobic peptide 3 in the absence of 0.5% acetic acid suggests a significant contribution of acetic acid in activity previously observed for hydrophobic peptides.
The effect of acetic acid on the growth of T. interdigitale
Inhibition of growth of T. interdigitale acetic acid was evaluated by means of experiments that determine the growth of fungi, as described in Materials and Methods. Culture of T. interdigitale were grown either raw or processed 3 different concentrations of acetic acid is islote at 30°C for 96 hours (11). This illustrates the lack of significant effect of 0.5% acetic acid, the same concentration as used for peptides 1-4 as solvent. This experiment, together with the lack of activity of peptide 3 in the absence of acetic acid suggests that peptide 4 is the most active compound against Trichophyton spp.
The effect of poly-L-lysine on growth of T. interdigitale
Because peptide 4 is visokoaktivniy peptide containing residues lysine and arginine, the antifungal activity of poly-L-forms of these amino acids was tested against T. interdigitale using analyses of growth inhibition, as described in Materials and Methods, in the absence of acetic acid. Received untreated control culture of T. interdigitale and culture, containing 1 mg/ml to 50 μg/ml of molecules of poly-L-lysine, ranging in length 27-100 100-200 residues and residues. The growth of T. interdigitale in each culture was assessed after 96 hours at 30°C. the Molecule poly-L-lysine both sizes inhibited growth of T. interdigitale (Fig), but, although the molecule is larger inhibited growth at all tested concentrations, the inhibitory activity with the molecule length 27-100 amino acids was observed only at higher concentrations (Fig). This suggests that ratingaverage effects of lysine on Trichophyton spp. depend on the size of the RA, and dose-dependent.
The effects of poly-L-arginine and poly-L-lysine on growth of T. rubrum
Antifungal activity of poly-L-arginine in comparison with poly-L-lysine was then tested against T. rubrum. Inhibition of growth was determined as in Materials and Methods, in the absence of acetic acid. T. rubrum was grown in the same environment, in an environment containing poly-L-arginine (length 28-86 amino acids) and poly-L-lysine (length 100-200 amino acids). Also put the control medium without inoculum. The cultures were maintained and growth monitoring was carried out for 96 hours at 30°C. As poly-L-arginine and poly-L-lysine inhibited growth of T. rubrum (Fig). Poly-L-arginine was more active in their inhibitory effects against T. rubrum than poly-L-lysine when tested at equivalent doses, a total of inhibiting growth at 1 mg/ml (Fig).
Inhibition of T. interdigitale and T. rubrum poly-L-arginine
The growth inhibition of Trichophyton spp. poly-L-arginine was tested using experiments that determine the growth of fungus, as described in Materials and Methods. Culture of T. rubrum and T. interdigitale were grown either in one environment, or in a medium containing 3 different concentrations of poly-L-arginine. Acetic acid was not present in any of the samples. Controls have been used to illustrate the background absorption of the medium. The growth of the fungus was determined by OD as described above, after 96 hours incubative 30°C (Fig). It is seen that polyalanine more active against both fungi up to 0.55 mg/ml (Fig).
The effect of reduced concentration (100 µg/ml) polyalanine against T. rubrum and T. interdigitale
The growth inhibition of Trichophyton spp. poly-L-arginine was tested using experiments that determine the growth of fungus, as described in Materials and Methods. Culture of T. rubrum and T. interdigitale were grown either untreated or treated with one concentration polyalanine (100 μg/ml), and acetic acid was not present in any of the samples. Controls have been used to illustrate the background absorption of the medium. The growth of the fungus was determined by OD as described above, after 96 hours of incubation at 30°C (Fig). Reduced concentration leads to loss of activity that illustrates the dose effect of polyalanine on Trychophyton spp.
The effect of peptide trimers (3 amino acids) on the growth of T. rubrum
Tested the activity of the peptide trimers poly-L-lysine, poly-L-arginine, poly-L-histidine and poly-L-tryptophan on the growth of T. rubrum. The growth inhibition was performed as in Materials and Methods, and T. rubrum were left either untreated or subjected to 2 mg/ml of each of the trimers. The cultures were maintained for 96 hours at 30°C. fungal Growth was measured by OD and the results were expressed in percentage of growth in untreated culture (Fig). Poly-L-arginine was moststrong peptide against T. rubrum, and required only a 3-amino acid polypeptide to cause a significant reduction in the growth of T. rubrum.
The effect of peptide 4 (1.2 mg/ml) and NaCl on the growth of T. interdigitale
Investigated the effects of different salt concentrations on the antifungal activity of the peptide 4 in respect of T. interdigitale. Analysis of inhibition of growth of T. interdigitale was performed as in Materials and Methods, in the absence of acetic acid. Cultures were left untreated or were exposed to the effect of peptide plus 4 number of NaCl concentrations from 100 mm to 500 mm. Culture of T. interdigitale maintained for 96 hours at 30°C and growth was assessed by OD as described above (Fig). On the antifungal activity of the peptide 4 did not influence the salt concentrations close to physiological conditions or in excess of their (Fig). Antimicrobial activity of endogenous β-defensins well described as inhibiting even low concentrations of salts.
The effect of peptide 4 against Candida albicans at high concentrations of salt
Survival of C. albicans was assessed as described in Materials and Methods, after 2 h of incubation at 37°C with a range of concentrations of peptide 4. Two concentrations of NaCl were introduced into the growth environment, to ensure the impact of physiological and very high salt concentrations (known as inhibiting peptide activity of endogenous β-defensins). Significant Kil the policy activity of peptide 4 was observed even at very high salt concentrations (Fig). With increasing concentration of peptide 4 suggests that the effect of higher salt concentration decreases (Fig). Therefore, the fungicidal activity of peptide 4 is not inhibited by salts.
The activity of poly-L-lysine, poly-D-lysine and poly-D-arginine against Candida albicans
Antifungal activity of poly-L-arginine compared to lysine and poly-L - compared to poly-D-lysine were evaluated to determine which of these peptide variants demonstrates improved activity against Candida albicans. Candida spp. incubated as described in Materials and Methods, for 2 hours at 37°C in the presence of 100 μg/ml, 1 mg/ml and 10 mg/ml poly-D-lysine, poly-L-lysine and poly-L-arginine. Survival was assessed as described above and demonstrated enhanced the antifungal activity of poly-L-arginine compared with poly-L-lysine (Fig). Also demonstrated that poly-D-lysine has a very similar antifungal activity with poly-L-lysine.
|A non-exhaustive list of bacterial pathogens of man|
|Group A, B, C, D, F, G, R|
|Type a, β, γ|
|pallidum||Including subspecies Pertenue & endemicum|
|Table 1 (continued)|
|A non-exhaustive list of fungal pathogens of man|
|td align="left"> Glabrata|
|Cryptococcus||neoformans||Bap neoformans and gattii and grubii|
|Emmonsia||Parva||Bap parva and crescens|
|Filobasidiella||neoformans||Bap neoformans and gattii|
|Histoplasma||Capsulatum||Bap capsulatum, dubiosii and farcinimosum|
|Trichophyton||Rubrum||Including var nigricans and granular|
|Mentagrophytes||Including var interdigitale and goetzii|
|Table 1 (continued)|
|A non-exhaustive list of parasitic pathogens of man|
|Table 1 (continued)|
|Non ecyuiui list of enveloped viral human pathogens|
|Enveloped virus: a virus with urine outer bilayer acquired through the separation membrane of the host cell|
|The alpha viruses:|
|Eastern encephalomyelitis of horses||Fever, malaise, headaches, encephalitis|
|Western encephalomyelitis of horses||Fever, malaise, headaches, encephalitis|
|Venezuelan encephalomyelitis of horses||Fever, malaise, headaches, encephalitis||Includes virus/fever, Mucambo, the Everglades, Tonate, Pixuna, Cabassou|
|River Ross||Fever, rash, arthralgia|
|Chikungunya||Fever, rash, arthralgia, arthritis|
|Mayaro||Fever, rash, arthralgia|
|O Nong-Niang||Fever, rash, arthralgia|
|Sindbis||Fever, rash, arthralgia||Fever Ockelbo, Karelian and Churchyard|
|Yellow fever||Hepatitis with hemorrhagic diathesis||High fever, chills, headache, muscle pain, vomiting and back pain, shock, bleeding, and kidney and liver failure|
|Dengue (types 1, 2, 3 and 4)||Immunopathological disease (shock and haemorrhage)||Dengue fever or Dengue haemorrhagic fever|
|Encephalitis St. Louis||Encephalitis|
|Encephalitis Murray valley||Encephalitis|
|Russian spring-summer encephalitis||Encephalitis||Tick|
|Disease kiesanowski forest||Encephalitis||Tick|
|The tick-borne encephalitis Powassan||Tick|
|West Nile fever|
|Kum is Inga||Tick|
|Scottish encephalomyelitis of sheep||Tick|
|Tat is Gina||Fever|
|Bunyamwera||Fever||Bunyamwera, Germiston, Ilesa, Shokwe, Tense, Wyeomyia|
|Fever||Apeu, Caraparu, Ithaca, Madrid, Marituba, Surucucu, Nepoui, Orinoco, Ossa, Reston|
|California||Fever or encephalitis||California encephalitis, Guaroa, Inkoo, La Crosse, rabbits, hares, Jamestown Canyon, Tahyna|
|GUAM||Fever||Kathu, GU is mA|
|Phlebotomy fever||Fever||Alenquer, Candino, Chagres, Naples, Punto Toro, rift valley fever, Sicily, Tuscany|
|Crimean-Congo fever||Hemorrhagic fever|
|Nirenska hemorrhagic fever||Fever|
|Hantaan||Hemorrhagic fever with renal syndrome or Hantavirus pulmonary syndrome||Hantaan (Korean hemorrhagic fever), Puumala, Seoul, Sin Nombre (Canyon Muerto), new York city, Black Creek Canal|
|LCM-Lassa-virus complex (arenavirus Old world)||Ippy, Lassa fever, lymph choriomeningitis, Mobile, Mobaa|
|Tacaribe-viral complex||Ampari, Flexal, Guanarito, Junin, Latin, Machupo, paraná, Pichinde, Sabia, Tamiami|
|Flu||Types a, b and C|
|Parainfluenza||From mild, febrile common cold to severe, potentially life-threatening croup, bronchiolitis and pneumonia||Types 1-4 (4 is divided into a and b)|
|Respiratory syncytial virus||Types a and b|
|SARS||Severe acute respiratory syndrome|
|European Lyssavirus bats|
|Vesiculovirus||Vesicular stomatitis||Types -4|
|T-cell leukemia human||Lymphoproliferative and neurological disorders||Types 1, 2 and 5|
|The herpes virus associated with Kaposi's sarcoma|
|The human immunodeficiency virus||AIDS||Types 1 and 2|
|Human immunodeficiency virus monkeys|
|Human foamy virus|
|The HERPES viruses|
|Herpes simplex||Genital and oral herpes||Types 1 and 2|
|The zoster virus||Chickenpox|
|Cytomegalovirus||Hepatosplenomegaly, retinitis, rash and involvement of the Central nervous system and possibly mononucleosis|
|The virus of Epstein-Barr||Mononucleosis|
|Human herpesvirus||Sudden exanthema (roseola)||Types 6 and 7|
|The Kaposi sarcoma virus||Kaposi's sarcoma||Man is ical herpesvirus type 8|
|Ebola Reston||Multiple hemorrhagic manifestations, significant involvement of the liver, disseminated intravascular coagulation and shock|
|Amino acid polymer codes|
|KKK - trimer L-lysine|
|RRR is a trimer of L-arginine|
|IUU - trimer L-histidine|
|WWW - trimer L-tryptophan|
|pK3-14 - poly-L-lysine-HBr 500-2000 Yes (3-14 A.K.)|
|pK7-27 - poly-L-lysine-HBr 1000-4000 Yes (7-27 A.K.)|
|pK100-200 - poly-L-lysine-HCl 15000-30000 Yes (100-200 A.K.)|
|pKd27-100 - poly-D-lysine-HBr 4000-15000D Yes (27-100 A.K.)|
|pR28-86 - poly-L-arginine-HCl 5000-15000 Yes (28-86 A.K.)|
1. The use of the peptide or pharmaceutically acceptable salt of formula I:
where the peptide contains from 3 to 200 amino acids, and where l and m represent integers from 0 to 10; n is an integer from 1 to 10; X and Y, which may be identical or different, represent a cationic amino acid selected from arginine and lysine, in the manufacture of medicines to treat fungal infections.
2. The use according to claim 1, where X and/or Y is a D or L-amino acids.
3. The use according to claim 1, where X and Y represented Aut an alternating amino acids.
4. The use according to claim 1, where X and Y are equal.
5. The use according to claim 1, where X represents lysine and Y is arginine.
6. The use according to claim 1, where the peptide contains from 3 to about 100 amino acids.
7. The use according to claim 1, where the peptide contains from 3 to 50 amino acids.
8. The use according to claim 7, where the peptide contains from 3 to 15 amino acids.
9. The use of claim 8, where the peptide contains from 3 to 7 amino acids.
10. The use according to claim 1, the peptide consists of from 3 to 200 amino acids.
11. The use according to claim 1, where the fungal infection is selected from the group consisting of tinea, histoplasmosis, blastomycosis, and aspergillosis.
12. The use according to claim 1, where a fungal infection is nedersetting infection.
13. The application of section 12, where a fungal infection is a yeast infection.
14. Use item 13, where a fungal infection caused by fungal pathogens of the genus Candida spp.
15. The use according to claim 1, where a fungal infection is dermatophyte infection.
16. The application indicated in paragraph 15, where the infection is caused by a fungal pathogen of the genus Trichophyton spp.
17. The application of article 16, where the fungal pathogen is a Trichophyton interdigitale.
18. The application of article 16, where the pathogen is a Trichophyton rubrum.
19. The application of clause 15, where a fungal infection is a skin infection.
20. The application of claim 19, where the infection is predstavljaet a onychomycosis.
21. Antifungal composition comprising a pharmaceutically effective amount of one or more peptides or their pharmaceutically acceptable salts and pharmaceutically acceptable carrier, excipient or diluent, where the specified peptide corresponds to the formula I:
where the peptide contains from 3 to 200 amino acids, and where l and m represent integers from 0 to 10; n is an integer from 1 to 10; X and Y, which may be identical or different, represent a cationic amino acid selected from arginine and lysine.
22. Antifungal composition according to item 21, containing at least two peptide of formula I, where these peptides are different.
23. Antifungal composition according to item 21, where excipient or diluent selected from the group consisting of acid, dimethyl sulfone, N-(2-mercaptopropionyl)glycine, 2-n-nonyl-1,3-dioxolane and ethyl alcohol.
24. Antifungal composition according to item 23, where excipient or diluent is an acid selected from the group consisting of acetic acid, citric acid, boric acid, lactic acid, propionic acid, phosphoric acid, benzoic acid, butyric acid, malonic acid, malic acid, oxalic acid, succinic acid or tartaric acid.
25. Antifungal composition according to the .24, where the acid is an acetic acid.
26. Antifungal composition according A.25, where acetic acid is represented in the composition at a concentration of less than 1, 0,5, 0,25, of 0.1, 0.05, or 0.01% of acetic acid.
SUBSTANCE: invention relates to agonists of neuropeptide-2 receptor with formula (I): Y-R1-R2-X-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-NH2(l), as well as their pharmaceutically acceptable salts, derivatives and fragments, in which substitutes assume values given in the description.
EFFECT: compounds and pharmaceutical compositions containing said compounds can be used to treat diseases which are modulated by neuropeptide-2 receptors, mainly obesity.
13 cl, 10 dwg, 1 tbl, 74 ex
SUBSTANCE: there is prepared identification peptide with amino acid sequence: Gly-Pro-Ala-Pro-Gln-Pro-Asp-Glu-Asp-Leu-Lys-Arg-Gln. The prepared peptide is applied to identify, purify and recover the recombinant proteins containing it. For making the recombinant proteins with a polypeptide label, an expression vector pDED is applied. Said vector contains a nucleotide sequence coding amino acid sequence of the identification protein.
EFFECT: invention allows extending range of the methods to identify the recombinant proteins.
14 cl, 6 dwg, 1 tbl, 6 ex
SUBSTANCE: present invention refers to medicine and concerns methods and compositions for gastroenteric upset therapy. Substance of the invention involves purified polypeptide of 14 amino acids for gastroenteric upset and condition therapy (including, e.g. gastroenteric motor disturbance, functional gastroenteric disease, gastroesophageal reflux disease (GERD), Crohn's disease, ulcerative colitis, inflammatory bowel disease. Additionally the invention involves method of guanylate cyclase activity improvement with using peptide specified above.
EFFECT: advantage of invention consists in extended application.
9 cl, 6 ex, 10 dwg
SUBSTANCE: invention concerns new depsipeptide compounds, as well as pharmaceutical compositions of these compounds and application of the compounds as antibacterial compounds.
EFFECT: methods of obtaining the new depsipeptide compounds and intermediary products applied in obtaining these compounds.
31 cl, 3 tbl, 25 ex
SUBSTANCE: method of obtaining dodecapeptide of the formula I: H-Asp-His-Leu-Asp-Lys-Gln-Thr-Gln-Thr-Pro-Lys-Thr-OH and tripeptide of the formula II: X-Asp(Y)-His-Leu-OH is the intermediate compound in its synthesis. Solid-phase synthesis of dodecapeptide I is realised by sequential growth of the peptide chain, beginning with the C-end dipeptidilpolymertill the obtaining of C-end nonapeptidilpolimer, which is condensed with the protected N-end tripeptide of the formula II: X-Asp (Y)-His-Leu-OH where X, Y are protected groups and the obtained dodecapeptidilpolymer is processed with an unblocking agent for removing the protective groups and the polymeric matrix and in 1 stage the end product is given out by means of HELC.
EFFECT: increasing the output of the end product and simplification of the process.
3 cl, 2 ex
FIELD: medicine; pharmacology.
SUBSTANCE: essence of the invention includes an agent for external application which structure includes a peptide from the allopherones group, allostatin-1 and auxiliary substances. The offered agent is applied to treatment of diseases of a skin and mucosa caused by a virus of herpes, a virus of papilloma, and also cosmetic defects of a skin.
EFFECT: scope expansion.
4 cl, 1 dwg, 4 tbl
SUBSTANCE: invention concerns biologically active compounds with inhibition effect on binding antigen with II class MHC molecules, particularly with II class MHC molecule HLA-DR2. Also, the invention proposes pharmaceutical compositions containing such compounds, and their application in obtaining medications for treatment or prevention of diseases connected to T-cell proliferation, such as autoimmune diseases and disorders of inhibition of binding II class MHC molecule HLA-DR2.
EFFECT: obtaining pharmaceutical composition for treatment or prevention of diseases connected with T-cell proliferation.
17 cl, 4 tbl, 6 ex
FIELD: medicine; pharmacology.
SUBSTANCE: invention refers to methods of self-specific T-cell vaccine production. Self-specific T-cell vaccine for disseminated sclerosis treatment includes attenuated T-cells which are reactive relative to one or several epitopes, yet SEQ ID NOS: 1-6 contain these epitopes. Invention is intended for treatment of autoimmune diseases, such as disseminated sclerosis or rheumatoid arthritis using self-specific T-cell vaccines. Besides, invention provides diagnostics of diseases associated with T-cells. Advantage of this invention implies that it can be applied for production of T-call vaccines with heterogenous gene VR-Dp-JR to take into consideration clonal shift if self-reactive T-cells.
EFFECT: method has improved efficiency.
10 cl, 7 ex, 2 dwg
FIELD: medicine, biotechnology.
SUBSTANCE: invention proposes variants of antibodies showing specificity to peptide domain located by both side of hinged site R76S77 in pro-BNP(1-108). Indicated antibodies recognize specifically also circulating pro-BNP(1-108) in human serum or plasma samples but they don't recognize practically peptides BNP(1-76) or BNP(77-108). Also, invention describes variants of peptides used in preparing antibodies. Amino acid sequence is given in the invention description. Also, invention discloses methods for preparing indicated antibodies and among of them by using indicated peptides. Also, invention describes methods for preparing antibody-secreting hybridoma, and hybridoma is disclosed prepared by indicated method. Also, invention describes a monoclonal antibody secreted by hybridoma 3D4 and deposited at number CNCM I-3073. Also, invention discloses variants for diagnosis of cardiac insufficiency in vitro and by using antibodies proposed by the invention. Also, invention describes a set used for detecting pro-BNP(1-108) in a biological sample. Using this invention simplifies detection of pro-BNP(1-108) circulating in human serum or plasma samples and provides specific detection of pro-BNP(1-108) that can be used in early diagnosis of human cardiac insufficiency.
EFFECT: valuable medicinal properties of antibodies.
24 cl, 16 dwg, 5 tbl, 20 ex
FIELD: organic chemistry, pharmacy.
SUBSTANCE: invention relates to an improved chelate conjugates of the formula: wherein each R1, R2 and R3 represents independently R group; Y represents -(A)n-X-Z wherein X represents -NH-, -CO2-, -N(C=O)-; Z represents a biological group for directed delivery that is chosen from 3-20-membered peptide, substrate for enzyme, enzyme inhibitor or synthetic compound binding with receptor; -(A)n represent linker wherein each A represents independently -CR2-, -NRCO-, -CONR-, -CR2OCR2 or polyalkylene glycol group; n represents a whole number from 2 to 10; each R group represents independently hydrogen atom (H) or (C1-C10)-alkyl, or two R groups in common with atoms to which they are added form (C3-C6)-carbocyclic saturated ring. Complexes with radioactive metals are useful as radioactive pharmaceutical preparation, especially, with 99mTc.
EFFECT: valuable medicinal properties of conjugates.
30 cl, 3 tbl, 4 dwg, 30 ex
SUBSTANCE: invention discloses novel synthetic RGD-like peptides capable of dose-dependant inhibition of thrombocyte aggregation.
EFFECT: obtaining novel compounds capable of dose-dependant inhibition of thrombocyte aggregation.
2 tbl, 1 ex
SUBSTANCE: invention relates to biotechnology and is a peptide which induces killer T cells ex vivo and which has an amino acid sequence as shown in one SEQ ID NOS: from 1 to 3. The disclosed peptide is used in an ex vivo agent for inducing anti-tumour immunity, in an ex vivo agent for inducing antigen-presenting cells, in an ex vivo agent which induces tumour-reactive T cells, as well as in an ex vivo pharmaceutical agent when treating or preventing tumours. The invention also relates to an antibody against the said peptide.
EFFECT: disclosed agents enable identification of glypican-3-derivative peptide, which can bond with HLA-A2, and activation of human killer T cells in order to provide an immunotherapy agent which may be effective in approximately 40% Japanese patients suffering from certain types of malignant tumours, accompanied by high level of GPC3 expression.
7 cl, 4 dwg, 1 tbl, 2 ex
SUBSTANCE: invention relates to contrast agents which contain a peptide vector linked with uPAR, marked with a visualising group.
EFFECT: obtaining a contrast agent for detecting urokinase plasminogen activator receptor.
6 cl, 6 ex
SUBSTANCE: invention relates to novel peptide compounds and their use in diagnostic optical visualisation techniques. More specifically, the present invention pertains to use of such peptide compounds as targeted vectors which are related to receptors associated with angiogenesis. The compounds are labelled using at least one cyanine reporter dye.
EFFECT: obtaining compounds which can be used as contrast agents in optical visualisation when diagnosing diseases related to angiogenesis.
9 cl, 5 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to bioengineering and specifically to obtaining biologically active substances of peptide nature, which have growth factor activity towards fibroblast proliferation and can be used in medicine. An oligopeptide of formula A-X1-X2-X3-X4-X5-B is obtained through in silico construction, where A is F; X1 is E, or Q, or S; X2 is N, or Q, or A, or G; X3 is K, or R, or T; X4 is K, or E, or is absent, X5 is K, or L, or is absent and B is OMe - methyl.
EFFECT: invention enables obtaining an oligopeptide with transformation growth factor (TGF-β) and oncostatin M (OSM) towards fibroblast proliferation, and expansion of the range of effective therapeutic agents with wound-healing effect, which take part in closing wounds during inflammation and cicatrisation.
4 dwg, 2 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to bioengineering and specifically to obtaining biologically active substances of peptide nature, which have growth factor activity towards collagen synthesis stimulation and can be used in medicine. An oligopeptide of general formula A-X1-X2-X3-X4-X5-B (I) is obtained through in silico construction, where A is Ac - acetyl; X1 is G or A or is absent; X2 is P or I, or L, or V, or A; X3 is G; X4 is P or I, or L, or V, or A; X5 is G or A, or is absent and B is OMe - methyl.
EFFECT: invention enables obtaining an oligopeptide with acidic (aFGF) and transformation (TGF-β) growth factor activity towards stimulation of collagen biosynthesis, and expansion of the range of effective therapeutic agents with wound-healing effect, which speed up regeneration of damaged tissue and cicatrisation.
4 dwg, 2 ex
SUBSTANCE: invention is related medicine and concerns applications of antibodies specifically recognising any prevailing variants of beta-amyloid peptide, Aβ40 and Aβ42, in preparation of a drug applied for prevention and-or treatment of Alzheimer's disease.
EFFECT: invention provides prevention of progression or reduction of symptoms, and/or decrease in amyloid deposition in an individual when administering an immunostimulating dose of peptide or specific antibody.
7 cl, 3 ex, 2 dwg
SUBSTANCE: small peptides of formula X1-X2-X3-X4-X5-X6-X7-R1, containing 7-12 amino acid residues are proposed.
EFFECT: said peptides are MC4 receptor agonists and are therefore useful in treating obesity and related diseases.
31 cl, 2 tbl, 82 ex
SUBSTANCE: present invention concerns a compound representing a selective agonist of a melanocortin-4 receptor of formula: Ac-Arg-c(Cys-D-Ala-His-D-Phe-Arg-Trp-Cys)-NH2 (SEQ ID NO:50) and its pharmaceutically acceptable salts, pharmaceutical compositions and methods for application thereof in preparation of drugs.
EFFECT: higher effectiveness of compound application.
20 cl, 8 dwg, 4 tbl, 4 ex
SUBSTANCE: invention relates to low-molecular derivatives of peptides which are used for preparing a pharmaceutical agent which inhibits laminin/nidogen reaction.
EFFECT: increased effectiveness of compounds.
2 cl, 12 dwg, 2 tbl, 30 ex
SUBSTANCE: there is offered a method to assess of enzyme's ability to the level of phosphorylation of polypeptide that implies a reaction of the analysed enzyme and a substratum presented with a biotin-conjugated fragment of 516 to 777 residues of a human insulin 1 receptor substratum (hIRS-1-p30), binding of the reaction product and immobilised streptavidin and detection of the level of phosphorylation by antibodies specific to the phosphorylated polypeptide residues.
EFFECT: according to the invention, the method allows identifying tyrosine and serine proteinases and can be taken as a basis of a test system for new modulators of their activity.
9 cl, 8 dwg, 4 ex