CN103394074A - Cyclic antimicrobial peptides - Google Patents
Cyclic antimicrobial peptides Download PDFInfo
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- CN103394074A CN103394074A CN2013102995185A CN201310299518A CN103394074A CN 103394074 A CN103394074 A CN 103394074A CN 2013102995185 A CN2013102995185 A CN 2013102995185A CN 201310299518 A CN201310299518 A CN 201310299518A CN 103394074 A CN103394074 A CN 103394074A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/64—Cyclic peptides containing only normal peptide links
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4723—Cationic antimicrobial peptides, e.g. defensins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The present invention relates to cyclic cationic peptides and their use in the treatment of microbial infections.
Description
The application be that December in 2006 21 days, application number are 200680053281.0 the applying date, denomination of invention divides an application for the application for a patent for invention of " cyclic antimicrobial peptides ".
Invention field
The present invention relates to ring-type cationic peptide and the application in the treatment infected by microbes thereof.
Background of invention
Antimicrobial peptide (AMP) has formed the basis of eukaryote immunity, and the first line of defence of opposing microbial destruction skin and mucomembranous surface is provided.The example of natural AMP comprises alexin and cathelicidin peptide family.The length of these AMP, sequence and configurations, but their topmost common ground are that molecule is little, band cation net charge and both sexes structure.Also from various bacteria, fungus, plant,, cation antimicrobial peptide little without isolating spinal animals and spinal animals, so it seems that antimicrobial peptide also in procaryotic defence, brought into play effect.
Natural AMP shows the broad spectrum of activity of resisting gram-positive bacteria and gram negative bacteria, yeast, fungus and enveloped virus.Pathogenic microorganism be it seems not to be had to obtain the resistance to these cationic peptides, and similarly, AMP has conservatively experienced the evolution of several thousand as important natural immunity host defense molecule.Therefore be understood that, AMP has become the potential goal in research for the therapeutic agent of multi-infection.Yet AMP is faced with technical challenge, and it is high in the recombination system production cost, has potent chemotaxis and inflammatory biological function, and these have all hindered natural AMP becomes therapeutic agent.
In our common pending application, we have shown is rich in some alkaline residue, as lysine or arginic linear peptides, has antimicrobial acivity, particularly antifungal activity.Yet, still also need to can be used in the medicine for the treatment of and prophylaxis of microbial infection.
Summary of the invention
According to a first aspect of the invention, wherein provide and comprised 2 to approximately 200 D and/or the amino acid whose peptide of L-, described aminoacid can be identical or different, is selected from hydrophobic amino acid and/or cationic amino acid, and peptide wherein is ring-type.This cyclic peptide can comprise 3 to approximately 100 D and/or L-aminoacid, and for example 3 to 50 D and/or L-aminoacid comprise 4 to approximately 50 D and/or L-aminoacid.
Peptide of the present invention can be for prevention and treatment infected by microbes.
Because the effectiveness of cyclic peptide of the present invention is high, stable to Proteolytic enzyme, insensitive in fact to salt, without liver toxicity, without haemolysis and be easy to synthesize, so they are hopeful as therapeutic agent.
The cationic charge of peptide of the present invention is considered to promote the polar head group of peptide on microbial film to be combined.By cyclisation, charged group is stablized with the form of crypto set (confirmation) more, thereby believed such anti-microbial effect that this attraction strengthens peptide that strengthened.
In aspect the present invention more specifically, provide the amino acid whose peptide that comprises according to formula I:
((X)
l(Y)
m)
n (I)
Wherein l and m are 0 to 10 integer, but l and m can not be 0; N is 1 to 10 integer; X and Y can be identical or different, for being selected from the aminoacid of hydrophobic amino acid and/or cationic amino acid, and are cyclic peptide as this peptide of drug use.
Peptide can comprise 2 to 50 aminoacid, and for example 3,4,5,6 or 7 until 50 aminoacid comprise that 3,4,5,6 or 7 until 10,15,20,25,30,35,40,45 or 50 aminoacid.
In preferred aspects of the invention, peptide comprises 2 to 15 aminoacid, for example 3 to 15 aminoacid.Preferably, peptide comprises 5 to 13 aminoacid.Further preferably comprise 3 to 7 aminoacid, for example 7 amino acid whose peptides.
Just s known as technical staff, mainly, according to chemistry and the physical property of amino acid side chain, aminoacid can be divided into to different kinds.For example, some aminoacid are considered to hydrophilic or polar amino acid usually, other be considered to hydrophobicity or nonpolar amino acid.Hydrophobic amino acid can be selected from the hydrophobic amino acid group that comprises glycine, leucine, phenylalanine, proline, alanine, tryptophan, valine, isoleucine, methionine, tyrosine and threonine; Cationic amino acid can be selected from ornithine, histidine, arginine and lysine.Term " hydrophobicity " and " cationic " that the present invention uses refer to that hydrophobicity is more than or equal to-1.10 and/or according to Fauchere and Pliska Eur.J.Med Chem.10:39,1983 described methods, and net charge is more than or equal to 0 aminoacid.Hydrophobicity or nonpolar amino acid also can refer to the aminoacid with uncharged non-polar sidechain under physiological pH, and this side chain is usually incompatible with aqueous solution.Aminoacid can be natural or synthetic.
In preferred aspects of the invention, X and/or Y are the cationic amino acid that is selected from histidine, ornithine, arginine and lysine.Preferred X and/or Y are arginine or lysine.
X and/or Y can be the hydrophobic amino acid that defines of the present invention or the optical isomer of cationic amino acid, as D or L-aminoacid.Preferred X and/or Y are D-aminoacid.
In preferred aspects of the invention, the peptide shown in formula I comprises at least 90%, and for example at least 95%, as 97-99%, or 100% D-aminoacid even.
In preferred aspects of the invention, the peptide shown in formula I comprises at least 90%, and for example at least 95%, as 97-99%, or 100% L-aminoacid even.
The present invention also comprises above-mentioned amino acid whose known isomers (constitutional isomer, stereoisomer, conformation and configurational isomer); peptide mimics (peptidomimetics) and analog, and (for example post translational modification) of natural modifications or chemical modification, include but not limited to phosphorylation, glycosylation, sulfonylation and/or hydroxylated above-mentioned aminoacid.
In general, peptide of the present invention does not comprise following aminoacid: aspartic acid, glutamic acid, and agedoite, glutamine or serine, even but these aminoacid exist, and some peptide of the present invention still can have biological activity.
Of the present invention more preferably aspect in, X and Y are identical.Preferably, X and Y are identical and are lysine and arginine.
In the peptide shown in formula (I), l and m can be 1,2,3,4,5,6,7,8,9 or 10, and n can be 1,2,3,4,5,6,7,8,9 or 10.
In the peptide shown in formula (I), l can be 1, n can be 1 and m can be between 4 and 9, for example m can be 3,4,5,6,7,8 or 9.
In the peptide shown in formula (I), l, n and/or m can be between 1 and 5, and for example 1,2,3,4 or 5.
In the peptide shown in formula (I), l and m can be the integers between 0 and 7, and n can be the integer between 1 and 10.
In the peptide shown in formula (I), l and m can be 0,1 or 2, and n can be the integer between 1 and 10.
In the peptide shown in formula (I), X and Y can be identical, and 1 can be that 0, m can be 1, and n can be 3,4,5,6,7,8,9 or 10.
In the peptide shown in formula (I), X and Y can be identical, and l and m can be 1, and n can be 2,3,4 or 5.
In the peptide shown in formula (I), X and Y can be identical, and l can be that 1, m can be 2, and n can be 1,2,3 or 4.
In the peptide shown in formula (I), X and Y can be identical, and l and m can be 2, and n can be 1,2,3 or 4.
In aspect the present invention more specifically, provide the amino acid whose cyclic peptide that comprises according to formula II:
(X)
n (II)
Wherein X and n are as mentioned above.Preferred X is lysine, arginine and ornithine.Preferred n is the integer between 3 and 15.
In one embodiment of the invention, X is arginine.
In alternative embodiment of the present invention, X is lysine.
In other alternative embodiment of the present invention, X is ornithine.
Peptide of the present invention can comprise one or more cysteine residues, for example reaches 6 cysteine residues, as 1,2,3,4,5 or 6 cysteine residues.
In addition, thereby the aminoacid sequence of peptide can be modified the variant that forms peptide, and this variant comprises that at least one amino acid residue in peptide substitutes another amino acid residue, comprises and uses substituting of D type rather than L-type.
One or more amino acid residues of peptide can be replaced by another, to change, to strengthen or keep the biological activity of peptide.Such variant can have and for example have at least the approximately biological activity of 10% corresponding unmanifest body peptide.Often use conservative amino acid, namely replace above-mentioned have similar chemistry and the aminoacid of physical property.Therefore, for example the conservative amino acid replacement can comprise lysine replacement arginine, ornithine or histidine; Perhaps arginine is replaced lysine or isoleucine, and ornithine is replaced histidine; Perhaps a kind of hydrophobic amino acid is replaced another kind of.After replacing, the biologic activity of screening variant.
With regard to common term, the term " peptide " that the present invention uses refers to a plurality of amino acid residues that couple together by peptide bond.This term can with peptide and protein Alternate, and same meaning.
In one embodiment of the invention, cyclic peptide is selected from:
K-K-K-K-K-K-K
R-R-R-R-R-R-R
The peptide that peptide of the present invention is normally synthetic.Peptide can be separate, the peptide of purification or their variant, these can be external synthetic, for example by the solid-phase peptide synthetic method, by enzymatic peptide, synthesize or by recombinant DNA technology.
In aspect the present invention more specifically, provide the method for preparation according to peptide of the present invention, the method comprises that reaction by peptide and coupling agent is by the peptide cyclisation shown in formula (I) or formula (II).
Coupling agent can be any can be between two ends (C-terminal and N-terminal) amino acid residue of the peptide of linear forms, for example between two amino acid backbones or side chain, form the reagent of peptide bond.On the selection of coupling agent, can affect the efficiency of coupling, thereby affect the output of cyclic peptide.Table 1 has shown coupling agent used in method of the present invention, and the technical staff will recognize that other known coupling agent also can be for the present invention.Preferred coupling agent is HATU-O-(7-azo BTA-1-yl)-N, N, N', N'-tetramethylurea hexafluorophosphoric acid ester.
Preferably, the reaction between peptide and coupling agent is to occur in alkali to exist in situation.Alkali can include, but are not limited to N-methylmorpholine (NMM) or diisopropylethylamine (DIEA).Preferably, reaction occurs under alkaline pH, and for example pH is between 8.5-9.Before peptide and coupling agent reaction, peptide can be modified into to the peptide that comprises blocking group.Blocking group can comprise: Pbf(2,2,4,6; 7-pentamethyl Dihydrobenzofuranes-5-sulfonyl), the tBu(tertbutyl ether), Mtr(methoxy trimethylbenzene sulfonyl) and, Pmc(2; 2,5,7,8-pentamethyl benzo dihydropyran-6-sulfonic acid chloride); Mbh(4,4-dimethoxy-benzyl hydride, 4; 4-dimethyloxybenzylhydride), Tmob(2,3; 4-trimethoxy benzyl), the Aloc(allyloxycarbonyl), the Fmoc(9-fluorenylmethyloxycarbonyl) and the Boc(tertbutyloxycarbonyl).After the reaction with coupling agent, can remove blocking group by the cracking under solutions of weak acidity, for example under the existence of trifluoracetic acid (TFA) solution.
In the skeleton cyclization process of peptide, the peptide of linear peptides C-terminal contacts with the activated group of coupling agent, and, along with reaction is carried out, on this amino acid whose α carbon, forms keto-enol formula intermediate.Therefore, when from ortho position carbon, removing activated group, enol (enol) (or enol, alkenol) intermediate can cause the formation of two kinds of enantiomers, and has formed peptide bond.The coupling agent one that passes through to use is activated, this racemization, be the formation of the diastereomer of the formation (for example dextrorotatory form and levorotatory form (namely being respectively d and l isomer)) of single amino acid enantiomer and peptide as a whole, just can occur on the cyclisation position.Owing to wishing that peptide of the present invention is pure enantiomer, therefore should reduce or avoid the generation of undesirable diastereomer.In order to reduce and avoid the generation of this diastereomer, and the peptide of producing pure diastereomer, peptide of the present invention can be modified to the peptide that comprises the achirality part, and this part has been avoided the racemization of peptide in the cyclization process.
Therefore of the present invention more preferably aspect in, the peptide of peptide of the present invention or method of the present invention definition is modified to and comprises the part of avoiding forming in cyclization process the diastereomer of peptide." racemic peptide " that the present invention uses referred to before the peptide cyclisation, comprises the optical isomer separately of a certain amount of (normally equivalent) PEPC end amino acid, for example the peptide of dextrorotation and left-handed form (namely being respectively d and/or l isomer).The part that is introduced into peptide is generally achirality aminoacid, can be aminoacid or the amino acid analogue of natural formation.Achirality aminoacid can be selected from glycine, Beta-alanine, 3-alanine, 4-Aminobutanoicacid, 5-aminovaleric acid and 6-aminocaprolc acid.In one embodiment of the invention, at C-terminal, peptide is modified into and comprises achirality aminoacid, for example glycine.
Except as mentioned above peptide of the present invention being modified into to C-terminal, comprise a part, the ratio of two kinds of enantiomer that form in the cyclization process of peptide depends on several factors, as solvent, cyclization time (incubation time) and the temperature of using in cyclization process (namely with the coupling agent reaction), and be used to promoting the activated group of cyclisation.
The invention still further relates to the peptide of the cyclisation that can obtain by method of the present invention.
In one embodiment of the invention, cyclic peptide comprises the aminoacid sequence that is selected from following amino acid sequences:
K-K-K-K-K-K-K
R-R-R-R-R-R-R
O-O-O-O-O-O-O
DR-DR-DR-DR-DR-DR-DR
DO-DO-DO-DO-DO-DO-DO
DK-DK-DK-DK-DK-DK-DK
In order to identify that undesirable toxicity to mammalian cell is very little or there is no the bioactive peptide of undesirable toxicity, can prepare single peptide or peptide storehouse (libraries of peptides), and can screen antimicrobial acivity and the toxicity of the peptide in single peptide or these peptide storehouses, include, but are not limited to antifungal, antibacterium, antiviral, protozoacide, Antiparasitic Activity and toxicity.
Peptide of the present invention can exist with multi-form, as with free acid, free alkali, ester and other prodrug, salt or tautomer, for example, and present invention includes all variant forms of these compounds.
Therefore, present invention includes salt or the prodrug of peptide of the present invention or peptide variant.
Peptide of the present invention can be with the form administration of pharmaceutically acceptable salt.Pharmaceutically acceptable salt of the present invention can be synthetic by parent peptide by the conventional chemical method, and this parent peptide comprises alkalescence or acidic moiety.In general, these salt are by at water or in organic solvent or both mixture, and are with stoichiometric suitable alkali or acid reaction, prepared by the peptide of organic acid or alkali form; Normally, to resemble ether, ethyl acetate, ethanol, isopropyl alcohol or acetonitrile be preferred to non-aqueous media.The list of the salt that is suitable for sees Remington's Pharmaceutical Sciences, 17th ed., and Mack Publishing Company, Easton, Pa., US, 1985, p.1418, therefore introduce the content of its disclosure as a reference; " the Handbook of Pharmaceutical Salts Properties Selection and Use " that also compiles referring to Stahl etc., Verlag Helvetica Chimica Acta and Wiley-VCH, 2002.
Therefore, present invention includes the pharmaceutically acceptable salt of peptide of the present invention, wherein the salt pair of the acid by preparing parent compound or alkali its modify, described salt is conventional nontoxic salts or the quaternary ammonium salt that formed by inorganic or organic acid or alkali for example.the example of such acid-addition salts comprises acetate, adipate, alginate, aspartate, benzoate, benzene sulfonate, disulfate, butyrate, citrate, camphorate, camsilate, cipionate, digluconate, lauryl sulfate, esilate, fumarate, glucose enanthate (glucoheptanoate), glycerophosphate, Hemisulphate, enanthate, caproate, hydrochlorate, hydrobromate, hydriodate, the 2-isethionate, lactate, maleate, malonate, mesylate, the 2-naphthalene sulfonate, nicotinate, oxalates, palmitate (palmoate), pectate, persulfate, the 3-phenpropionate, picrate, Pivalate, propionate, succinate, tartrate, rhodanate, toluene fulfonate and undecylate.Basic salt comprises ammonium salt, alkali metal salt such as sodium salt and potassium salt, and alkali salt such as calcium salt and magnesium salt, contain the salt of organic base such as two cyclohexylamine salt, N-methyl D-glutamine, and the salt that contains aminoacid such as arginine, lysine, etc.In addition, nitrogenous basic group can be quaternized by following reagent, and lower halogenated alkane, as chloromethanes, ethyl chloride, chloropropane, chlorobutane, Celfume, bromoethane, N-Propyl Bromide, n-butyl bromide, iodomethane, iodoethane, iodopropane and iodobutane; Dialkyl sulfate, as Dimethylsulfate, diethyl sulfide hydrochlorate, dibutyl sulfide hydrochlorate and diamyl sulfate; Long-chain halogenide, as chloro decane, bromodecane, iodo decane, lauryl chloride, lauryl bromide, lauryl iodine, myristyl chlorine, myristyl bromine, myristyl iodine, stearic chlorine, stearyl bromine and stearyl iodine; Halogenated aromatic alkane, as bromobenzene methane and bromobenzene ethane and other.
Can prepare by the carboxylate of the variant of peptide of the present invention or peptide, namely by peptide, with the target alkali of monovalent or many equivalents, contact in a conventional manner, and metal hydroxides alkali for example, as sodium hydroxide; Metal carbonate or bicarbonate, as sodium carbonate or sodium bicarbonate; Perhaps amine alkali, as triethylamine, triethanolamine etc.
The present invention includes the prodrug for the pharmaceutically active form of described peptide; for example wherein one or more functional groups are protected or derivatization; but it can be converted into functional group in vivo; as be converted in vivo the carboxylate of free acid, maybe can be converted into the situation of the protected amino of free amine group.The term " prodrug " that the present invention uses refers in particular to the structure that is converted into rapidly in vivo precursor structure, for example in blood, is hydrolyzed.
The present invention aspect more specifically provides and has comprised the pharmaceutically peptide of the present invention of effective dose, or the pharmaceutical composition of two or more different peptides of the present invention.
Compositions also comprises pharmaceutically acceptable carrier, excipient and diluent.The phrase " pharmaceutically acceptable " that the present invention uses refers in rational medical judgment scope, be suitable for the mankind's tissue and contact, perhaps with the tissue of animal, contact in some cases, and do not produce overdosage toxicity, stimulation and anaphylaxis or other problem or complication, and have those compounds, raw material, compositions and/or the dosage form of the reasonable benefit/risk ratio that matches.
Peptide of the present invention is particularly useful as antimicrobial peptide, for example antibacterium, fungus, yeast, parasite, protozoacide and virus.The present invention can use term " antimicrobial peptide " to define any peptide with microbicidel and/or bacteriostatic activity, and comprise described any have antibacterium, antifungal, antifungal, parasiticide not exclusively, protozoacide, antiviral, infection, infection and/or sterilization, kill algae, kill ameba, microbicidel, kill antibacterial, antifungal, parasiticide, kill the peptide of protozoacide character.
Aspect preferred, the invention provides the purposes of peptide according to the present invention in the medicine for the preparation of the treatment infected by microbes.
" infected by microbes " refers to by antibacterial, parasite, protozoacide, virus or fungus and comprises the infection that yeast etc. causes." pathogen " is generally defined as any diseases induced organism.
Bacterial pathogen can derive from and be selected from following group, but is not limited to the bacterial species of following group, comprising: staphylococcus, staphylococcus aureus (for example staphylococcus aureus NCTC10442) for example, staphylococcus epidermidis; Chlamydiaceae, chlamydia trachomatis for example, Chlamydia pneumoniae and chlamydia psittaci; Enterococcus, for example enterococcus faecalis; Streptococcus pyogenes; The Li Site Pseudomonas; Rhodopseudomonas; Mycobacterium, for example mycobacterium tuberculosis; Enterobacter; Campylobacter; Salmonella; Streptococcus, for example A or B group streptococcus, streptococcus pneumoniae; Helicobacterium, for example helicobacter pylori; Neisseria, Diplococcus gonorrhoeae for example, Neisseria meningitidis; Borrelia burgdoyferi; Shigella, for example shigella flexneri; Escherichia coli (Escherichia coli O 157: H7NCTC12900); Haemophilus, for example hemophilus influenza; Francisella tularensis; Bacillus, for example anthrax bacillus; Clostridium, for example bacillus botulinus; Yersinia's genus, for example Yersinia pestis; Treponema; Burkholderia, Burkholderia cepacia for example, glanders burkholderia and Burkholderia Pseudomallei.
In preferable use according to the present invention, bacterial pathogens is staphylococcus aureus and escherichia coli.
Viral pathogens can derive from and be selected from following group, but is not limited to the virus of following group, comprising: HIV (human immunodeficiency virus) (HIV1 type and 2 types); Human T-leukemia virus's (HTLV1 type and 2 types); Ebola virus; Human papillomavirus (for example HPV-2, HPV-5, HPV-8, HPV-l6, HPV-18, HPV-31, HPV-33, HPV-52, HPV-54 and HPV-56); Papovavirus; Rhinovirus; Poliovirus (polioviras); Herpesvirus; Adenovirus; Epstein-Barr virus; Influenza virus; Hepatitis B virus and hepatitis C virus, smallpox virus, rotavirus or sars coronavirus.
The parasitic disease substance can derive from and be selected from following group, but is not limited to the parasite of following group: trypanosoma (schizotrypanum cruzi and trypanosoma bocagei), leishmaniasis, Giardia, Trichomonas, Entamoeba, Naegleria, Acanthamoeba, Schistosoma, Plasmodium, Cryptosporidium (Crytosporidium spp.), Isospora, Balantidium, Loa loa, ascarid, heart worm belongs to, and toxoplasma is toxoplasma gondii for example.
In preferable use according to the present invention, infected by microbes is fungal infection.
fungal pathogens can derive from and be selected from the subordinate, but be not limited to the fungus (comprising yeast) with the subordinate: Candida (for example Candida albicans), Epidermophyton, the mould Pseudomonas of outer blank handle, Microsporon, Trichophyton (for example trichophyton purpureatum and trichophyton interdigitalis), tinea belongs to (Tinea spp.), Eurotium, Blastomyces, Blastoschizomyces (Blastoschizomyces spp.), Coccidioides, Cryptococcus (for example Cryptococcus histolyticus), Histoplasma, Paracoccidioides (Paracoccidiomyces spp.), Sporothrix, Absidia, Cladophialophora belongs to, Fonsecaea, Phialophora, Lacazia belongs to, Arthrographis belongs to, Acremonium chrysogenum belongs to, the madura actinomyces, Apophysomyces belongs to, Haplosporangium, group's Basidiomycotina, Beauveria, Chrysosporium, Conidiobolus, Cunninghammella, the crescent Pseudomonas, Geotrichum, Graphiem, Leptosphaeria (Leptosphaeria spp.), the mould Pseudomonas of horse traction plug (for example pityrosporum horse traction plug mycete), Mucor, Neotestudina, Nocardia, Nuo Kaer soil Pseudomonas, paecilomyces, Phoma, Piedraia, Pneumocystis belongs to, pseudo-Allescheria, pyrenomycetes belongs to (Pyrenochaeta spp), Rhizomucor, Rhizopus, Rhodotorula, Saccharomycodes, Scedosporium, the mould genus of broom, Sporobolomyces, Syncephalastrum, trichoderma, Piedraia, Ulocladium (Ulocladium spp.), Ustilago, Verticillium Nees, Wangiella (Wangiella spp.).
In preferable use according to the present invention, fungal pathogens belongs to Trichophyton or Cryptococcus.For example fungal pathogens can be trichophyton purpureatum, trichophyton interdigitalis or Cryptococcus histolyticus.
Fungal infection can be general, local, subcutaneous, skin or mucosal infections.
Although some non-dermatophytes, also can cause skin infection as yeast, the infection of the local fungal of skin and finger (toe) first is normally caused by dermatophytes.Cutaneous fungal infection can comprise that tinea infects, tinea barbae (beard) for example, tinea capitis (head), tinea corporis (health), tinea cruris (groin), facial tinea (face), the tinea manuum (hands), tinea pedis (foot), tinea unguium (referring to (toe) first), tinea versicolor (pityriasis), tinea incognito and black tinea.These infection can be caused by the fungus of Epidermophyton, Microsporon and Trichophyton (trichophyton purpureatum and trichophyton interdigitalis).
Cutaneous fungal infection can be the infection of skin, hypothallus (lamina), horny layer, first (fingernail and toenail) or hair.What mention especially is the cutaneous fungal infection that the dermatophytes by Trichophyton, Epidermophyton or Microsporon causes.the example of dermatophytes comprises acrothesium floccosum, microsporum canis, the little spore of Ao Duni is mould, microsporum gypseum, microsporum nanum, Microsporum ferrugineum, microsporum distortum, the little spore of yellowish-brown is mould, trichophyton purpureatum, Trichophyton mentagrophytes mutation trichophyton interdigitalis, Trichophyton mentagrophytes mutation tuberosity Trichophyton mentagrophytes (Trichophyton mentagrophytes var.nodulare), trichophyton tonsurans, the Sudan Trichophyton (Trichophyton Soudanese), trichophyton violaceum, Trichophyton megninii (Trichophyton megnini), She Enlai mattress Trichophyton, trichophyton gallinae, the krajdenii Trichophyton, the yaoundei Trichophyton, trichophyton equinum, erinacei Trichophyton and trichophyton verrucosum.
In particular of the present invention, cutaneous fungal infection is tinea unguium.Term " tinea unguium " includes but not limited to, position, distal side first mo(u)ld bottom half, shallow vindication colour pattern, near-end white first mo(u)ld bottom half, secondary dystrophic, former dystrophic, the endonyx type, beads bacterial type (for example tinea unguium and chronic mucocutaneous disease) tinea unguium and tinea unguium.
The non-dermatophytes fungus relevant with tinea unguium comprises Eurotium, Cephalosporium, sharp Fusariumsp, scopulariopsis brevicaulis (Scopularis brevicaulis), the mould Pseudomonas of post.
Peptide of the present invention is to the equal effective antimicrobial peptide of multiple Pathogenic organisms.In addition, peptide of the present invention also can include but not limited to the symptom relevant with mucosal infections for the treatment of other symptom, cystic fibrosis for example, the infection of gastrointestinal tract, genitourinary system, urinary system (renal infection or cystitis) or respiratory system.
Peptide of the present invention also can be used for the treatment of or prevent the usually infection relevant to skin, and comprising wound, ulcer and damage, for example skin trauma is as incised wound or burn, and the symptom of being correlated with.
In aspect the present invention is preferred, peptide is used for the treatment of bacterial skin infection or " pyoderma ".
Term " treatment " relates to peptide of the present invention effect aspect being benefited the patient who makes to stand (infectivity) disease misery, comprises the symptom of improving the patient or delays disease progression.
The term " trauma care " that the present invention uses can comprise wound healing and relevant situation, and the treatment that promotes, increases or accelerate organization healing, comprise the rear scar scar formation of operation, burn, ulcer, psoriasis, accelerate tissue remodeling (tissue remodelling), for example plastic operation or organ transplantation are postoperative.
Therefore, the substrate that provides peptide of the present invention to use or adhere in aspect the present invention more specifically.Preferred substrate is suitable for being applied to wound or carries to wound site.Preferred substrate makes peptide of the present invention be transferred to wound surface from substrate, to realize its antibiotic effect.Substrate can be dressing, for example wound dressing.Dressing can comprise fibrous material, or can be collagen sample material.
Peptide of the present invention also can or be used in disinfectant as disinfectant.In this case, peptide or pharmaceutical composition of the present invention can be separately or with other disinfectant combined administration in pending surface." pending surface " that the present invention uses can be substrate or the medical treatment device that the present invention defines.
In aspect more specifically, the invention provides the method for the treatment of or prevention experimenter infected by microbes, comprising give described experimenter treat effective dose according to peptide of the present invention.
In a preferred method of the invention, infected by microbes is fungal infection.In the method for the invention, peptide is locally applied to described experimenter's skin or refers to (toe) first.
Peptide of the present invention, compositions and method can be treated mammal, birds and other animal.These mammals and birds comprise the people, Canis familiaris L., and cat and domestic animal, as horse, cattle, sheep, goat, chicken, turkey etc.In addition, peptide of the present invention, compositions and method also can be treated plant.
When the experimenter was animal, method of the present invention can be applied to the position with finger (toe) first sample feature, includes but not limited to hoof, pawl and foot.
Except the peptide treatment, method of the present invention can comprise can strengthen peptide to the treatment that refers to the infiltration of (toe) first.This realizes by chemistry and physical method.Naturopathy for example refers to (toe) first etching (nail etching), or finger (toe) the first back layer of rasping away, and can strengthen the permeability of peptide of the present invention.The infiltrative chemical method of finger (toe) first that strengthens peptide of the present invention is to refer to that by destruction physics or chemical bond in the keratin of (toe) deck realize.Refer to that (toe) onychomalacia agent includes but not limited to carbamide and salicylic acid, its enhancing refers to that the hydration of (toe) first refers to the density of (toe) first with reduction, therefore can improve the permeability of peptide of the present invention.The compound that comprises mercapto groups can cracking refer to the disulfide bond in (toe) first keratin, and can cause the permeability of stabilization removal and raising medicine.
In aspect more specifically, the invention provides the method for the treatment of experimenter wound, comprising to wound administering therapeutic effective dose according to peptide of the present invention or substrate.
In order to obtain desirable effect, peptide and variant thereof or combination can be with single dose or divided dose administrations, for example at least about 0.01mg/kg to about 500-750mg/kg, at least about 0.01mg/kg to about 300-500mg/kg, at least about 0.1mg/kg to about 100-300mg/kg or at least about 1mg/kg to about 50-100mg/kg body weight, perhaps at least about 1mg/kg to 20mg/kg body weight, although other dosage also can provide useful effect.Dosage will change along with various factors, include but not limited to selected peptide and clinical effectiveness thereof, disease, body weight, health, health status, mammiferous age, what will realize is treatment or prevention, and whether peptide passes through chemical modification.
According to the present invention, giving therapeutic agent can be single dose, multiple dose, continuous or mode intermittently, and this depends on for example receiver's physiological situation, and the purpose of administration is treatment or prevention, and the known other factors of professional and technical personnel.Give peptide of the present invention continuous during predetermined time in fact, or can be the form with the dosage at a series of intervals.Local and whole body administration is all taken into account
In order to prepare compositions, can synthetic peptide or otherwise obtain peptide, with expectation, carry out purification as required, then through lyophilizing and stabilisation.Then regulate peptide to suitable concentration, and randomly with other agent combination.The absolute weight of the particular peptide that comprises in unit dose can vary widely.For example, can give approximately 0.01 to about 2g or approximately 0.01 to about 500mg peptide at least a of the present invention, or specificity is for the multiple peptide of specific cells classification.Alternatively, unit dose can, from about 0.01g to about 50g, from about 0.01g to about 35g, from about 0.1g to about 25g, from about 0.5g to about 12g, from about 0.5g to about 8g, change from about 0.5g to about 4g or from about 0.5g to about 2g.
Therefore, one or more suitable unit dosage forms that comprise therapeutic peptide of the present invention can pass through the number of ways administration, comprise oral, intestinal outer (comprising subcutaneous, intravenous, intramuscular and intraperitoneal), rectum, skin, transdermal, intrathoracic, in lung, intranasal (breathing) approach.Therapeutic peptide also can be mixed with lipid formulations or for slow release (for example using microencapsulation, referring to WO94/07529 and U.S. Patent No. 4,962,091).If be suitable for, dosage form can be discrete unit dosage forms so that use, and can prepare by any method that pharmaceutical field is known.These methods can comprise therapeutic agent and liquid-carrier, solid matrix, semi-solid carrier, finely divided solid carrier or their combined hybrid, and as required product is added subsequently after required induction system or molding to the step that enters required induction system.
When therapeutic peptide of the present invention is made into for oral administration, they usually with pharmaceutically acceptable carrier, diluent or excipient composition to form pharmaceutical preparation or unit dosage forms.For oral administration, peptide can be to be rendered as powder, granule, solution, suspension, emulsion, or be in natural or synthetic polymer or resin for from chewing gum, taking in active component.Bioactive peptide also can be rendered as pill, electuary or paste.The therapeutic peptide liquid of the present invention of oral administration can be formulated into slow release formulation, and for example peptide can or be placed in the device that other slow release is carried by coating, microencapsulation.In such preparation, the active component total amount accounts for the 0.1-99.9% of weight of formulation.
The pharmaceutical preparation that comprises therapeutic peptide of the present invention can use the composition of knowing and easily obtaining by the method preparation of this area.For example, peptide can be mixed with tablet, capsule, solution, suspension, powder and aerosol etc. together with the excipient of commonly using, diluent or carrier.The example that is suitable for excipient, diluent and the carrier of these preparations comprises buffer agent and filler and extender, as the derivant of starch, cellulose, sucrose, mannitol and silicon.Also can comprise binding agent, as carboxymethyl cellulose, hydroxy methocel, hydroxypropyl methylcellulose and other cellulose derivative, alginate, gelatin and polyvinylpyrrolidone.The wetting agent that can comprise such as glycerol, disintegrating agent such as calcium carbonate and sodium bicarbonate.Can also comprise the reagent that postpones stripping, as paraffin.Also can comprise again absorption enhancer (Resorption accelerators), as quaternary ammonium compound.Can comprise surfactant, as spermol, glyceryl monostearate.The absorption carrier that can add such as Kaolin and bentonite; The lubricant that can also comprise such as Talcum, calcium stearate and magnesium stearate, and solid polyethylene glycol; Also can add antiseptic.Compositions of the present invention also can comprise thickening agent, as cellulose and/or cellulose derivative.They also can comprise natural gum as xanthan gum, guar gum or carbo glue or arabic gum, or alternative Polyethylene Glycol, bentonite and montorillonite clay etc.
For example, the tablet or the capsule sheet (caplets) that comprise peptide of the present invention can comprise buffer agent, as calcium carbonate, magnesium oxide and magnesium carbonate.Suitable buffer agent also comprises acetate, citrate, borate and phosphate etc.Capsule sheet and tablet also can comprise non-active ingredient, as cellulose, pregelatinized Starch, silicon dioxide, hydroxypropyl methylcellulose, magnesium stearate, avicel cellulose, starch, Talcum, titanium dioxide, benzoic acid, citric acid, corn starch, mineral oil, polypropylene glycol, sodium phosphate, zinc stearate etc.Hard or the Perle that comprises at least a peptide of the present invention can comprise non-active ingredient such as gelatin, avicel cellulose, sodium lauryl sulphate, starch, Talcum and titanium dioxide etc., and liquid-carrier such as Polyethylene Glycol (PEGs) and vegetable oil.In addition, the casing capsule sheet or the tablet that comprise at least a or multiple peptide of the present invention be designed to prevent in the gastric disintegrate, and more neutrally in duodenum under the environment of alkalescence, dissolve.
Therapeutic peptide of the present invention also can be formulated into elixir or solution so that oral administration, or is mixed with the solution that is suitable for the intestinal external administration, for example intramuscular, subcutaneous, intraperitoneal or intravenous approach.The pharmaceutical preparation of therapeutic peptide of the present invention also can be adopted the form of aqueous solution or anhydrous solution or dispersion, or the form of alternative Emulsion, suspending agent or ointment.
Therefore, therapeutic peptide for the preparation of intestinal external administration (for example can be mixed with, by injection, for example the group annotates or continuous infusion), and can be present in the form of unit dose in ampoule, pre-filled syringe, small size infusion container or multi-dose container.Bioactive peptide and other composition can form suspension, solution or emulsion in oiliness or aqueous carrier, and can comprise regulator (formulatory agent), as suspending agent, stabilizing agent and/or dispersant.Alternatively, bioactive peptide and other composition can be powder types, and this is the aseptic separation by sterile solid, or by the carrier with being suitable for, the solution that before for example using, aseptic pyrogen-free water forms obtains through lyophilizing.
These preparations can comprise pharmaceutically acceptable carrier well known in the art (carriers), carrier (vehicles) and adjuvant.For example, can from the acceptable organic solvent of physiological viewpoint, prepare solution with one or more, outside dewatering, described solvent is selected from acetone, acetic acid, ethanol, isopropyl alcohol, dimethyl sulfoxide, glycol ether is as be called " Dowanol " product sold with name, polyethylene glycols and Polyethylene Glycol, C
1-C
4The Arrcostab of short chain acids, ethyl lactate or isopropyl lactate, the product that fatty acid triglycercide is sold as be called " Miglyol " with name, isopropyl myristate (isopropyl mytrisate), animal oil, mineral oil and vegetable oil and polysiloxanes.
The solvent or the diluent that comprise peptide of the present invention can comprise acid solution, dimethyl sulfoxide, N-(2-sulfydryl propionyl) glycine, 2-n-nonyl-l, 3-dioxolanes and ethanol.Preferred solvents/diluents is acid flux material, for example acetic acid, citric acid, boric acid, lactic acid, propanoic acid, phosphoric acid, benzoic acid, butanoic acid, malic acid, malonic acid, oxalic acid, succinic acid or tartaric acid.
Also considered to comprise the combination product of one or more peptides of the present invention and one or more other antimicrobial or antifungal, described other antimicrobial or antifungal be the polyenoid class for example, as amphotericin B, amphotericin B lipid complex (ABCD), AM Bison (L-ABM) and Nystatin liposome; Azole and triazole type, voriconazole, fluconazol, ketoconazole, itraconazole, posaconazole (pozaconazole) etc.; Glucan synthase inhibitors, as Caspofungin, MFG (FK463) and V-echinocandin (LY303366); Griseofulvin; Allylic amines, as terbinafine; Flucytosine or other antifungal, comprise those that the present invention describes.In addition, can consider that peptide and local antifungal combine, local antifungal such as ciclopirox olamine, haloprogin, tolnaftate, undecylenate (salt), the local Nystatin, amorolfine used, butenafine, naftifine, terbinafine and other topical remedy.
In addition, peptide can be mixed with slow release formulation etc.The composition of preparation can make said preparation can within the regular hour, discharge bioactive peptide, and for example the specific part at intestinal or respiratory tract discharges.Coating, peplos and protectiveness substrate can be by for example polymeric materials, as polylactic acid-glycollic acid, and liposome, microemulsion, microgranule, nano-particle or wax are made.These coatings, peplos and protectiveness substrate are for coating indwelling equipment, such as support, conduit, peritoneal dialysis catheter, pumping equipment (draining devices) etc.
For topical, as known in the art, active medicine can be mixed with and directly apply to target area.The form that is mainly topical application and prepares has adopted for example emulsifiable paste, emulsion, gel, powder, dispersion, microemulsion, the lotion of regulating thicker or rarelyr, the medicated cushion of dipping, ointment or rod, aerosol (for example spraying or foam), soap, detergent, the form of soap class lotion or soap blank.Other conventionally form for this purpose comprises medical dressing, coating binder (coated bandages), or other polymeric cover, ointment, emulsifiable paste, lotion, paste, gel, spray and aerosol.Therefore, can carry therapeutic peptide of the present invention with for percutaneous drug delivery by patch and binder.Alternatively, also peptide can be mixed with to sticky polymers, as the part of polyacrylate or acrylate/vinyl acetate co-polymer.In order to be suitable for prolonged application, may wish to use the substrate layer with micropore and/or ventilative, can make like this hydration of skin and dipping drop to minimum.Substrate layer can be anyly can provide required protection and the thickness of support function.Suitable thickness is generally approximately 10 to approximately 200 microns.
Topical can refer to the form of (toe) first coating or paint.For example, the antifungal peptide can be mixed with the solution for topical, wherein comprises ethyl acetate (NF), the aqueous isopropanol of isopropyl alcohol (USP) and poly-[methyl vinyl ether/maleic acid] mono—n—butylester.
The pharmaceutical preparation of topical application can comprise for example physiologically acceptable buffer salt solution, this solution comprises between about 0.001mg/ml and 100mg/ml, and for example one or more specificitys between 0.1mg/ml and 10mg/ml are directed to the indication that will be treated or the peptide of the present invention of disease.
For example, ointment and emulsifiable paste can uses or the oleaginous base preparation, wherein add suitable thickening agent and/or gellant.Lotion can use or the oleaginous base preparation, and usually also contains one or more emulsifying agents, stabilizing agent, dispersant, suspending agent, thickening agent or coloring agent.Bioactive peptide also can import to carry by electron ion, and for example United States Patent(USP) Nos. 4,140, and 122; Disclosed method in 4,383,529 or 4,051,842.The percentage by weight of the peptide of the present invention that exists in topical formulations will depend on many factors, but usually can be 0.01% to 95% of total formulation weight amount, and is typically 0.1-85% weight.
Drop, as eye drop or nasal drop, can adopt the therapeutic peptide in one or more aqueouss or non-aqueous substrate to prepare, and in this substrate, also comprises one or more dispersants, solubilizing agent or suspending agent.Liquid spray can drive with pump, or is convenient to from pressurized package, transporting out.Drop can, by simple eye with dropper bottle with cover (eye dropper-capped bottle), by being suitable for carrying the plastic bottle of liquid contents drop, or be carried by the given shape closed container.
Therapeutic peptide can further be mixed with for oral cavity and throat topical.For example, active component can be mixed with and further contain flavoured base, normally the lozenge of sucrose and arabic gum or tragakanta (lozenge); The lozenge (pastilles) that comprises the compositions in inert base such as gelatin and glycerol or sucrose and arabic gum; And the collutory that comprises the present composition in suitable liquid-carrier.
For the carrier of pharmaceutical preparation of the present invention and/or the specific non-limitative example of diluent, comprise water and physiologically acceptable buffer salt solution, as the phosphate buffered saline(PBS) of pH7.0-8.0.
Peptide of the present invention also can be through respiratory tract administration.With regard to sucking or be blown into administration, compositions can adopt dry powder form, for example therapeutic agent and suitable powdery substrate such as the pulverulent mixture of lactose or starch.When the form administration with aerosol or suction, therapeutic peptide of the present invention also can be with the aqueous solution administration.Therefore, other aerosol drug preparation can comprise for example physiologically acceptable buffer salt solution, in solution, comprise between about 0.001mg/ml and 100mg/ml, for example 0.1 and 100mg/ml between, as 0.5-50mg/ml, 0.5-20mg/ml, 0.5-10mg/ml, one or more specificitys of 0.5-5mg/ml or 1-5mg/ml are for the peptide of the present invention of the indication that will be treated or disease.
Description and claims of running through the application, word " comprises (comprise) " and the variant of " comprising (contain) " and these words, for example " comprise (comprising) " and " comprising (comprises) " means " including but not limited to ", and do not want to get rid of (and eliminating) other parts, additament, component, integer or step.
Run through description and claims of the application, odd number has comprised plural number, unless context separately has needs.Especially, when using indefinite article, explanation should be understood to consider plural number and odd number, unless context separately has needs.
The technical characterictic that is described together with the specific aspect of the present invention, embodiment or embodiment, integer, characteristic, compound, chemical group (chemical moieties) or group will be understood to the other side, embodiment or the embodiment that applicable to the present invention, describe, unless incompatible with it.
Embodiment
Materials and methods
Embodiment 1
The volume that equates by the protected peptide by 1eq and 1eq(eq=) HATU is dissolved in the DMF(dimethyl formamide with the concentration of 100mg/ml) in, make 7 amino acid whose PL200 peptides carry out cyclisation.In order to improve pH value, add the DIEA(diisopropylethylamine of 2.5eq), and by the HPLC tracking reaction process.When reaction completes, precipitation of peptides in water, and wash in addition.Then peptide is dry and remove to protect to form final cyclic peptide with Fluohydric acid..After this, before lyophilizing, use ion exchange chromatography to replace the Fluohydric acid. solvent with acetic acid.
Embodiment 2
By the protected peptide by 1eq and the NaHCO of 5eq
3The PyBOP of (sodium bicarbonate) and 2eq is dissolved in DMF with the concentration of 28.5mg/ml, makes 7 amino acid whose pR60 peptides carry out cyclisation.By the TLC following response, and complete precipitation of peptides in Shi Zaishui when reacting, and wash in addition.Then peptide is dry and remove to protect to form final cyclic peptide with Fluohydric acid..After this, before lyophilizing, use ion exchange chromatography to replace the Fluohydric acid. solvent with acetic acid.
Embodiment 3
The cyclisation of 7 amino acid whose pR60 peptides:
Solution 1: by the HBTU (Mwt=379.3 of 57mg, 0.15 mM) or the HATU (M=380.3 of 57mg, 0.15 mM) and the 0.92mg/ml NMM (n-methyl morpholine, Mwt=101.2,0.55 mM) of 60 μ l be dissolved in the DMF of 2.86ml.
Solution 2: by the H-[Arg of 288mg (Pbf)]
7-OH(Mwt=2877.6,0.1 mM) (7 amino acid whose pR60 peptides) be dissolved in the DMF of 0.71ml.
In 30 minutes, solution 2 is added drop-wise in solution 1.With wet pH reagent paper, check pH value, and pH value is necessary for 8.5-9.The stirred reaction mixture * that spends the night at room temperature.Mixture is through vacuum concentration.Add NaHCO
3(5%) solution.The precipitation of protected cyclic peptide after filtration and wash with water.Obtain 150-200mg.TFA/ water (95/5, v/v) in (the protected peptide 10ml of 1g), carry out the cracking of Pbf group.Enriched mixture also adds the thick product of IPE precipitation.Obtain the thick ring-type Arg of 100-110mg.
(* use that HATU carries out be coupled at 5 hours after complete.The amount of NMM depends on H-[Arg (Pbf)]
7Excessive TFA in-OH).
It is 95% enantiomer that synthetic cyclic peptide has purity at least, and trends towards changing between 97-99%.According to the synthetic cyclic peptide of the method, having purity at least is 95% enantiomer.
Meat soup dilution antifungal susceptibility test
Adopt the standard of research on standard institute of clinical laboratory (Clinical Laboratory Standard Institute, CLSI were NCCLS in the past) approval, determine the sensitivity of Relative Fungi bacterial strain to the peptide of cyclisation.Use the susceptibility of " reference method of the meat soup dilution antifungal susceptibility test of filamentous fungi, the standard M38-P of approval) " test fungus.Use the susceptibility of " reference method of the meat soup dilution antifungal susceptibility test of yeast, the standard of approval-second edition M27-A) " test yeast.
Meat soup dilution antibacterium susceptibility test
Adopt the standard of research on standard institute of clinical laboratory (CLSI was NCCLS in the past) approval, determine the sensitivity of Related Bacteria bacterial strain to the peptide of cyclisation.Use " reference method of the dilution antimicrobial susceptibility test of anaerobic growth antibacterial, the standard of approval-7th edition M7-A7) susceptibility of bacteria tested.
Hemolytic test
The peptide of studying is divided into to three parts with desired concn and is placed in the Nunc96 orifice plate, and carry out serial dilution (100 μ l) with 1:1.By human red blood cell (the RBC) (1x10 of 100 μ l through (50ml HBSS washing 3 times) gathering of washing
8RBC/ml) be added in instrument connection, and hatch 3 hours under 37 ℃.After hatching, then to the HBSS that adds 100 μ l in all holes, and by orifice plate 4 ℃ of lower overnight incubation.Shift out 100 μ l supernatant, and be placed in new microtitration plate, under 450/620nm, use Sunrise orifice plate readout instrument (Tascam) reading.Also comprised independent buffer, buffer and RBC, and water and RBC hole (establishing 4 multiple holes) in contrast.With Graph Pad(Prism software) data are drawn and carried out statistical analysis.
Result
The sequence of the peptide of cyclisation
The sequence of analyzed peptide is as follows:
Peptide 1: ring-type-K-K-K-K-K-K-K
Peptide 2: ring-type-R-R-R-R-R-R-R
Peptide 3: ring-type-K-K-K-K-K-K-K-G
Peptide 4: ring-type-R-R-R-R-R-R-R-G
Peptide 5: ring-type-O-O-O-O-O-O-O-G
Peptide 6: ring-type-DR-DR-DR-DR-DR-DR-DR-G
Peptide 7: ring-type-DO-DO-DO-DO-DO-DO-DO-G
Peptide 8: ring-type-DK-DK-DK-DK-DK-DK-DK-G
The synthetic middle aminoacid that uses that prefix " D " is illustrated in peptide is the D-isomer." O " represents the alpha-non-natural amino acid ornithine.
Synthesized the cyclic peptide that comprises 3,5,9,11,13 or 15 arginine or lysine, and after measured active (data do not show).
The antibacterial activity of the peptide of cyclisation
Peptide 1 is exposed in the culture of escherichia coli and staphylococcus aureus, after under 37 ℃, cultivating again 16 hours, for the MIC of two kinds of microorganisms, is 1mM(table 2).Under this concentration, peptide 1 has suppressed the growth of escherichia coli and staphylococcus aureus fully.
With peptide 2, repeat this experiment.2 couples of colibacillary MIC of peptide are 0.1mM; The MIC of 2 pairs of staphylococcus aureuses of peptide is 1.0mM.This peptide that shows cyclisation has significant effect to bacterial activity.
And the activity that the size linear peptides corresponding with peptide 1 and peptide 2 shows respectively is significantly lower than peptide 1 and peptide 2.
The antifungal activity of the peptide of cyclisation to trichophyton purpureatum
Trichophyton purpureatum is tested the susceptibility of peptide 1-8.The MIC to the trichophyton purpureatum culture that peptide 1 shows is 0.1mM(table 2).The MIC to the trichophyton purpureatum culture that peptide 2 shows is 0.25mM.
And the activity that the size linear peptides corresponding with peptide 1 and peptide 2 shows respectively is significantly lower than peptide 1 and peptide 2.
Peptide 3-8 has an independent glycine residue that is introduced into the cyclic peptide ring.Therefore, with respect to 7 aminoacid of peptide 1 and peptide 2, the length of peptide 3-8 is 8 aminoacid.The antifungal activity (MIC(mM) that peptide 3-6 shows anti-trichophyton purpureatum is respectively 4.0,2.0,4.0,1.0).Even peptide 7-8 does not demonstrate the antifungal activity of anti-trichophyton purpureatum yet under maximum detectable concentration (4mM).
Peptide 3-6 demonstrates antifungal activity, but the introducing of non-cationic aminoacid glycine has weakened antifungal activity significantly.For example, this can be from peptide 2(without glycine; MIC=0.2mM) with peptide 4(, add glycine; MIC=2.0mM) specific activity is found out (table 2).This data show length is that 7 and 8 amino acid whose cyclic peptides all have antifungal activity, but when the cationic of peptide reduces, the antifungal activity of trichophyton purpureatum is also reduced.
The inhibition of Trichophyton between the peptide zygodactyl of cyclisation
Between zygodactyl, Trichophyton is tested the susceptibility of peptide 2-8.The antifungal activity of the anti-trichophyton interdigitalis of peptide 2-8 sees table 2.Between peptide 2,4 and 6 zygodactyls, Trichophyton has activity.
The inhibition of the peptide of cyclisation to Cryptococcus histolyticus
Use the susceptibility of " reference method of the meat soup dilution antifungal susceptibility test of yeast, the standard of approval-second edition M27-A) " test Cryptococcus histolyticus to peptide 4 and 6-8.
Table 2 shows that cation cyclic peptide 4 and 6-8 have antifungal activity (MIC is respectively 1.0mM, 0.5mM, 2.0mM and 0.5mM) to pathogenicity yeast Cryptococcus histolyticus.
The antimicrobial acivity of 2 pairs of selected pathogenic microorganisms of peptide
The antimicrobial acivity that has shown 2 pairs of 60 kinds of selected pathogenic microorganisms of peptide in table 3.Just as can be seen, the Candida of antifungal, particularly dermatophytes, scopulariopsis brevicaulis, pityrosporum horse traction plug mycete, non-white and the strongest antimicrobial acivity of bacteria Escherichia coli (i.e. minimum MIC) have as one man been seen.
In the peptide of cyclisation, use the amino acid whose effect of enantiomer
Table 2 has shown the contrast situation of the inhibition of full L and full D ring-type cationic peptide.Peptide 4 and 6 is the full L of equivalent and the full D ring-type cationic peptides that comprise amino acids Arginine (7 aminoacid) and glycine (1 aminoacid).The antifungal activity of the anti-trichophyton purpureatum of full D type is better than full L-type (MIC be respectively 1.0 and 2.0mM).The antifungal activity of the complete anti-trichophyton interdigitalis of D type is better than full L-type (MIC be respectively 0.25 and 0.5mM).The antifungal activity of the complete anti-yeast Cryptococcus histolyticus of D type is better than full L-type (MIC be respectively 0.5 and 1.0mM).These activity that show the full D type of this peptide are better than full L-type.
Peptide 3 and 8 is the full L of equivalent and the full D ring-type cationic peptides that comprise amino acid lysine (7 aminoacid) and glycine (1 aminoacid).The antifungal activity of the anti-trichophyton purpureatum of full L-type is better than full D type (MIC is respectively 4.0 Hes > 4.0mM).Two kinds of peptides do not show the antifungal activity (MIC of two kinds of peptides is all greater than 4.0mM) of anti-trichophyton interdigitalis.
The hemolytic activity of the peptide of cyclisation
In concentration, surpass under the concentration that shows antifungal activity, the hemolytic activity of ring-type cationic peptide is negligible (table 4).
The liver toxicity of the peptide of cyclisation
Under the concentration of concentration close to the demonstration antifungal activity, peptide 2,9 and 10 does not demonstrate liver toxicity.
Table 1: coupling agent
(O type) (N-type) (O type) (N-type)
Urea salt/ammonium salt
HBTU:R
1=R
2=CH
3X=CH(N type structure)
HBPyU:R
1, R
2=(CH
2)
4X=CH(the unknown)
HATU:R
1=R
2=CH
3X=N(N type structure)
HAPyU:R
1, R
2=(CH
2)
4X=N(N type structure)
Imonium salt
BOMI:R
1=R
2=CH
3, R
3=H; X=CH(N type structure)
BDMP:R
1, R
3=(CH
2)
3, R
2=CH
3X=CH(N type structure)
DPMP:R
1=R
2=(CH
2)
4, R
3=Ph; X=CH(the unknown)
AOMP:R
1, R
3=(CH
2)
3, R
2=CH
3X=N(the unknown)
Phosphonium salt
BOP：R
1=R
2=CH
3；X=CH
PyBOP：R
1,R
2=(CH
2)
4；X=CH
AOP：R
1=R
2=CH
3；X=N
PyAOP：R
1,R
2=(CH
2)
4；X=N
Table 2: the antimicrobial acivity (MIC of the peptide of cyclisation to selected pathogenic microorganism; MM)
Table 3: the antimicrobial acivity (MIC of 2 pairs of selected pathogenic microorganisms of peptide; MM)
1The staphylococcus aureus of MRSA(methicillin resistance)
2Ref. No.: DM2006 517; DM2006 902; DM2006 932; DM2006 953; DM20061008; DM2006 1093; DM2006 1377
3The staphylococcus aureus of MSSA(methicillin-sensitivity)
4Ref. No.: 97.2935.K; 98.1695.K; 97.2637.D; 98.2028.X; 05.5240.R; 00.9523.R; 03.8996.T; 98.1515.F; 00.5472.R; 00.1039.P; 02.6225.E; 03.3200.J; 01.7995.S; 03.8951.G; 00.9521.M; 97.1636.D
Table 4: selected peptide is to erythrocytic hemolytic activity
| Peptide | Full test concentration (mM) | Haemolysis |
| 1 | ||
| 2 | 73,7 | Nothing |
| 3 | 10.0 | Nothing |
| 4 | 10.0 | Nothing |
| 5 | 10.0 | Nothing |
| 6 | 5.0 | Nothing |
| 7 | 1.0 | Nothing |
| 8 | 1.0 | Nothing |
Claims (12)
1. the purposes of cyclic peptide in the medicine that infects for the preparation of the treatment antibacterial, described cyclic peptide comprises the aminoacid according to formula I:
(X)
n (I)
Wherein, X is that arginine and n are the integers between 3 to 15.
2. purposes according to claim 1, wherein said peptide comprises 5 to 13 aminoacid.
3. purposes according to claim 2, wherein said peptide is comprised of 3 to 7 aminoacid.
4. purposes according to claim 3, wherein said peptide is R-R-R-R-R-R-R.
5. according to the described purposes of any one in aforementioned claim, it is caused by being selected from following antibacterial that wherein said antibacterial infects: staphylococcus, chlamydiaceae, Enterococcus, Li Site Pseudomonas, Rhodopseudomonas, Mycobacterium, Enterobacter, campylobacter, Salmonella, Streptococcus, Helicobacterium, neisseria, Borrelia burgdoyferi, Shigella, escherichia coli, haemophilus, Francisella tularensis, Bacillus, Clostridium, yersinia's genus, treponema and Burkholderia.
6. purposes according to claim 5, wherein said antibacterial is staphylococcus, escherichia coli, Rhodopseudomonas and Burkholderia.
7. purposes according to claim 6, wherein said antibacterial is staphylococcus aureus or escherichia coli.
8. according to the described purposes of any one in aforementioned claim, wherein said infection is selected from mucosal infections or symptom, gastrointestinal infection, urogenital infections, urinary system infection and the respiratory system infection relevant with it.
9. the described purposes of any one according to claim 1-7, wherein said infection is skin infection.
10. the described purposes of any one according to claim 1-7, it is used for the treatment of wound.
11. one kind comprises the pharmaceutically cyclic peptide of effective dose and the compositions of pharmaceutically acceptable supporting agent, excipient or diluent, described cyclic peptide comprises the aminoacid according to formula I:
(X)
n (I)
Wherein, X is that arginine and n are the integers between 3 to 15.
12. compositions according to claim 11, wherein said peptide is R-R-R-R-R-R-R.
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| GBGB0526120.1A GB0526120D0 (en) | 2005-12-22 | 2005-12-22 | Therapeutic peptides |
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| US77650506P | 2006-02-24 | 2006-02-24 | |
| US60/776505 | 2006-02-24 | ||
| CN2006800532810A CN101389642B (en) | 2005-12-22 | 2006-12-21 | Cyclic antimicrobial peptides |
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| CN2006800532810A Active CN101389642B (en) | 2005-12-22 | 2006-12-21 | Cyclic antimicrobial peptides |
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| CN (2) | CN103394074B (en) |
| BR (1) | BRPI0619641B8 (en) |
| DK (1) | DK1976863T3 (en) |
| ES (1) | ES2400734T3 (en) |
| GB (1) | GB0526120D0 (en) |
| NZ (1) | NZ569946A (en) |
| PT (1) | PT1976863E (en) |
| SG (1) | SG10201403126RA (en) |
| SI (1) | SI1976863T1 (en) |
| UA (1) | UA97633C2 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111018953A (en) * | 2020-01-19 | 2020-04-17 | 安徽农业大学 | Cyclocasein-isoleucin-leucin-chromo-threonine with antifungal and free radical scavenging activity and preparation method thereof |
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| SI3429616T1 (en) | 2016-03-15 | 2021-12-31 | Igor Polyakov | Compositions for the treatment and prevention of hoof and claw diseases |
| CN108314706B (en) * | 2018-01-25 | 2020-06-16 | 中国医学科学院医药生物技术研究所 | Polypeptide specifically bound with Ebola virus activated envelope glycoprotein and application thereof in resisting viruses |
| CN113264987B (en) * | 2021-05-31 | 2023-11-17 | 安徽农业大学 | Cyclic color-threo-valyl-isoleucyl-leucinyl peptide with antifungal and free radical scavenging activities and preparation method thereof |
| CN113307848B (en) * | 2021-05-31 | 2023-11-17 | 安徽农业大学 | Cyclic color-silk-valyl-isoleucyl-leucinyl peptide with antifungal and free radical scavenging activities and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995003327A2 (en) * | 1993-07-26 | 1995-02-02 | Biosynth S.R.L. | Peptides for neutralizing the toxicity of lipid a |
| US20050107289A1 (en) * | 2001-05-04 | 2005-05-19 | The Scripps Research Institute | Anti-microbial peptides and compositions |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5858974A (en) * | 1995-07-20 | 1999-01-12 | Xoma Corporation | Anti-fungal peptides |
| US7033991B2 (en) * | 2001-07-16 | 2006-04-25 | Board Of Supervisors Of Louisiana State University And Agriculture And Mechanical College | Inhibiting furin with polybasic peptides |
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2005
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2006
- 2006-12-21 BR BRPI0619641A patent/BRPI0619641B8/en active IP Right Grant
- 2006-12-21 CN CN201310299518.5A patent/CN103394074B/en not_active Expired - Fee Related
- 2006-12-21 DK DK06842187.4T patent/DK1976863T3/en active
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- 2006-12-21 SI SI200631545T patent/SI1976863T1/en unknown
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995003327A2 (en) * | 1993-07-26 | 1995-02-02 | Biosynth S.R.L. | Peptides for neutralizing the toxicity of lipid a |
| US20050107289A1 (en) * | 2001-05-04 | 2005-05-19 | The Scripps Research Institute | Anti-microbial peptides and compositions |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111018953A (en) * | 2020-01-19 | 2020-04-17 | 安徽农业大学 | Cyclocasein-isoleucin-leucin-chromo-threonine with antifungal and free radical scavenging activity and preparation method thereof |
| CN111018953B (en) * | 2020-01-19 | 2022-08-26 | 安徽农业大学 | Cyclocasein-isoleucyl-leucyl-tryptophyl-threo-peptide with antifungal and free radical scavenging activities and preparation method thereof |
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| Publication number | Publication date |
|---|---|
| CN101389642A (en) | 2009-03-18 |
| BRPI0619641B1 (en) | 2018-01-09 |
| GB0526120D0 (en) | 2006-02-01 |
| SI1976863T1 (en) | 2013-04-30 |
| UA97633C2 (en) | 2012-03-12 |
| BRPI0619641A2 (en) | 2012-12-11 |
| CN101389642B (en) | 2013-08-07 |
| ES2400734T3 (en) | 2013-04-11 |
| NZ569946A (en) | 2011-12-22 |
| DK1976863T3 (en) | 2013-03-04 |
| CN103394074B (en) | 2018-05-22 |
| PT1976863E (en) | 2013-03-05 |
| BRPI0619641B8 (en) | 2021-05-25 |
| SG10201403126RA (en) | 2014-10-30 |
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