CN101535337B

CN101535337B - Peptides

Info

Publication number: CN101535337B
Application number: CN200780040243.6A
Authority: CN
Inventors: R·纽
Original assignee: LEXCICON Ltd
Current assignee: LEXCICON Ltd
Priority date: 2006-09-20
Filing date: 2007-09-20
Publication date: 2014-05-14
Anticipated expiration: 2027-09-20
Also published as: CN101535337A; GB0618512D0

Abstract

An internally-constrained cyclic oligopeptide comprising a ring of at least six amino acids for specifically binding to a target ligand, wherein the ring comprises a plurality of amino acid domains, each domain comprising at least two epitope-forming amino acids, and two or more associating functional groups positioned so that they form one or more intra-cyclic associations; whereby the cyclic oligopeptide is constrained in a single conformation so that the epitope-forming amino acids form an epitope in each domain, each epitope being capable of specifically binding to a target ligand.

Description

Peptide

The present invention relates to a kind of inner affined annular oligopeptides, the pharmaceutical composition that comprises this oligopeptides, the application in medical science of this oligopeptides and pharmaceutical composition, and prepare the method for this oligopeptides.

Background of invention

Known protein acceptor is generally combined with its target ligands by epi-position, and epi-position is amino acid whose combination, forms the sub-fraction in protein macromolecule.

The protein acceptor of cell surface often produces signal through triggering in cell, causes and the another kind of protein binding that is called protein ligands.In part, be called epi-position with the part of acceptor interaction, conventionally combined by a few amino acids coming in close proximity to each other in peptide chain backbone.The example of epi-position be positioned at protein surface can with antibody or interactional those structures of φt cell receptor, but in fact, any region that protein surface can be identified by another kind of albumen all belongs to epi-position structure.Because the combination of protein receptor and epi-position may be the important step that disease pathogen is learned, or contrary, it is an important step for the treatment of disease, therefore identifying the functional group of formation epi-position may be a kind of fruitful approach to exploitation novel drugs, described new drug is the preparation containing in conjunction with the epi-position of this receptor.

Obstruction has two kinds of challenges with this method developing new drug thing.A kind of be need identify can be used as drug molecule, can in conjunction with corresponding acceptor with treatment disease epi-position.The second challenge is to design the molecule that can keep and provide the amino acid combination that forms this epi-position, thus the strong binding interactions of realization and cell receptor.

The authentication method that can form the amino acid combination of epi-position is known.In traditional combinational chemistry, what must consist of with different order synthetic hundreds of different groups (as amino acid) may combine, and detects effect separately, could identify the most promising sequence that can go out in conjunction with certain specific receptors.This kind method is consuming time, expense is high and be subject to the restriction of the chemical reaction character for heterogeneity is linked together.

WO 01/01140 provides a kind of improving one's methods of epi-position of identifying.Provide can with the composition of ligand interaction.Said composition comprises the multiple different conjugates that are assembled into by non covalent bond, and each conjugate contains a base and tail base.The tail base of conjugate forms hydrophobicity aggregate, each conjugate each other can free movement in assemblage, therefore in the time there is part, at least 2 stature bases by appropriate location form can with the epi-position of ligand interaction, its interaction strength is higher than a base independent situation separately.The multiple conjugate that evaluation has required biologic activity comprises: select one group of conjugate that is listed as, forms each other non-covalent connection containing a basic matrix, in this conjugate, tail base hydrophobicity is assembled and can move freely each other between conjugate, and whether detect interaction between non-covalent connection and part abundant.Utilize multiple this process of a basic matrix column weight of modifying to find the abundant interaction between non-covalent connection and part.This method by the epi-position that depends on base combination close to each other and produce identify can binding specificity acceptor most promising sequence, and synthesize hundreds of not isoplastic may combinations without the combinational chemistry with traditional.This method only depends on the epi-position that base combination close to each other produces.Just do not need synthetic other chemicals once synthesize one group of conjugate, only simple all conjugates mixing need be formed to different probes by non-covalent connection.

Although this new composition and method success for the identification of forming epi-position in conjunction with the functional group of target ligands, can form required epi-position but still need to provide, and improve to produce with the interactional stability of target ligands and specificity the improvement composition that biological answer-reply reacts.

Attempt the similar peptide of a preparation epi-position that comprises binding site by Amino acid profile, but usually met with failure because these peptides do not have the biologic activity identical with protein acceptor.

The binding site of protein is to be made up of the oligopeptides of the non-contiguous different piece of protein chain, once attempts rebuilding this kind of binding site by the free oligopeptide solution of composite liberation, but could not produce this active binding site.

Therefore, thus the object of the invention be to provide can form required epi-position, can be with the stability that improves and specificity and the target ligands improvement oligopeptides that generation biological answer-reply react that interacts.

Summary of the invention

The invention provides a kind of annular oligopeptides, the ring being formed by least 6 amino acid that it comprises energy specific binding target ligands, ring in this oligopeptides comprises multiple amino acid regions, at least 2 epi-position formative amino acid are contained in each region, and two or more associativity functional groups, its location can form in one or more rings and connect; This annular oligopeptides is constrained to single conformation, and the epi-position formative amino acid in each region forms epi-position, each epi-position energy specific binding target ligands.

The conformation that cyclic peptide is known in this area is more tied than linear oligopeptides.In cyclic peptide peptide end move freely limited.Because they together by chemical anchors.But cyclic peptide still has the flexibility of certain degree, the stable bond that makes them be not suitable for participating in target ligands interacts.

Structure contains two or more annular oligopeptides in conjunction with functional group, and its location can allow them form connection in one or more rings, and this annular oligopeptides is subject to be become single configuration by internal constraint.This makes the epi-position formative amino acid energy specific binding target ligands in each region, and its combination stability and specificity are more available higher than so far.Described epi-position formative amino acid can form stable epi-position, induces biological answer-reply to react with ligand interaction.Because the epi-position forming is like this stable, thereby is improved with the interaction of target ligands.

US 2005/0107289 discloses the anti-microbial agents and the composition that comprise cyclic peptide, the aminoacid sequence that described cyclic peptide contains D-alternately and L-a-amino acid or beta-amino acids composition.This article is also openly pointed out, thinks that this cyclic peptide can self be assembled into supramolecular structure or be combined with microbial film in the film of microorganism.This supramolecular structure can be for example nanotube.There is a hole in the pipeline center of each nanotube, round the peptide backbone of the cyclic peptide of a series of stacks.Ion and small molecules can see through the hole of this kind of nanotube.

US 2005/0107289 do not have to disclose its cyclic peptide whether contain epi-position formative amino acid and form can specific binding target ligands epi-position.US 2005/0107289 does not illustrate that constraint that whether its cyclic peptide encircled interior connection is to form this epi-position yet.

G.Abbenante etc. are " thiazole is with the controlling conformation of oxazoline ring to marine source annular octapeptide, the conformation of Novel chair shape and the large ring of ship shape " (Conformational Control by Thiazole andOxazoline Rings in Cyclic Octapeptides of Marine Origin.Novel MacrocyclicChair and Boat Conformations) J.Am.Chem.Soc.1996, 118, in 10384-10388 mono-literary composition, disclose Amino acid profile parts (Threonine, halfcystine) with conformation confinement ring (thiazole, oxazoline) adaptation with regulate ocean macromole (albumen) three-dimensional structure and reactive behavior.Annular octapeptide 1, c[Ile-Thr-D-Val-Cys-Ile-Thr-D-Val-Cyc-] there is very large flexibility, take many low-energy configurations.This cyclic peptide does not encircle interior connection, is not constrained for single conformation and forms multiple epi-positions.

G.Abbenante etc. are at " thiazole is with the controlling conformation of oxazoline ring to marine source annular octapeptide; the conformation of Novel chair shape and the large ring of ship shape " J.Am.Chem.Soc.1996,118, in 10384-10388 mono-literary composition, cyclic peptide 2 is also disclosed, c[Ile-Thr-D-(Val) Thz-Ile-Thr-D-(Val) Thz-], this peptide is shown as single false chair shape conformation in solution.Synthetic cyclic peptide 7, c[(Ile) Oxn-D-(Val)-Thz-(Ile) Oxn-D-(Val) Thz-] produce and be subject to highly constrained false ship shape or the large ring of the shape of a saddle.The annular octapeptide 8 that cyclic peptide 7 acid hydrolysiss produce is shown as the ship shape conformation with larger flexibility.The thiazole that these annular octapeptides are existed is with the constraint to a certain degree of/Huo oxazoline, but they can not form multiple epi-positions.

R.M.Cusack etc. are at " conformation of annular octapeptide and heterocycle constraint impact in conjunction with calcium on it " (Conformations of cyclic octapeptides and the influence of heterocyclic ringconstraints upon calcium binding) J.Chem.Soc., Perkin Trans.2, the thiazole 4 kind annular octapeptides different with oxazoline confinement ring number in heterocycle are disclosed in 2000,323-331, mono-literary composition.Peptide 1,2 and 3 has been taked different shapes in solution.The thiazole that these annular octapeptide amine are existed is with the constraint to a certain degree of/Huo oxazoline, but they can not form multiple epi-positions.

R.M.Cusack etc. are at " conformation of annular octapeptide and heterocycle constraint impact in conjunction with calcium on it " J.Chem.Soc., Perkin Trans.2,2000, in 323-331 mono-literary composition, also disclose and lacked thiazole with the peptide 4 of oxazoline ring, there is very large flexibility thereby this cyclic peptide of molecular model display lacks constraint except hydrogen bond, in solution, may take countless multiple conformations.Peptide 4 does not have in any ring and connects, and is not constrained for single conformation and forms multiple epi-positions.

Describe now annular oligopeptides of the present invention in detail.

Described annular oligopeptides contains the ring being made up of at least 6 amino acid, and this ring comprises multiple amino acid regions, and at least two epi-position formative amino acid are contained in each region, and two or more associativity functional groups.

In one embodiment, the ring that this annular oligopeptides contains 6 amino acid compositions, this ring comprises two amino acid regions, and two epi-position formative amino acid are contained in each region, and two associativity functional groups.In a preferred embodiment, this annular oligopeptides contains the ring being made up of 8 amino acid, and this ring comprises two amino acid regions, and three epi-position formative amino acid are contained in each region, and two associativity functional groups.

In another embodiment, this annular oligopeptides contains the ring forming with upper amino acid by 8.In this embodiment, described ring preferably comprises three associativity functional groups, forms in ring and connects and three amino acid regions of generation, and each region is containing three epi-position formative amino acid.

The amino acid using in this annular oligopeptides can be any natural amino acid, its substitutive derivative, analogue and its D-type amino acid.

In a preferred implementation of the present invention, three epi-position formative amino acid have three-dimensional chemical configuration alternately, i.e. L-D-L or D-L-D.This has special advantage, because can make the amino acid whose side chain of epi-position formative be orientated with plane configuration, all towards equidirectional.This make epi-position formative amino acid each other can be closely near forming epi-position.

In another embodiment, the ring that this annular oligopeptides contains 10 amino acid compositions, this ring comprises two amino acid regions, and 4 epi-position formative amino acid are contained in each region, and two associativity functional groups.This epi-position formative amino acid has three-dimensional chemical configuration identical or that replace, i.e. L-L-L-L, D-D-D-D, L-D-L-L, L-L-D-L, L-L-L-D, D-L-L-L, L-L-D-D, D-D-L-L, L-D-L-D, D-L-D-L, L-D-D-L, D-L-L-D, D-L-D-D, D-D-L-D, D-D-D-L or L-D-D-D.

The position of described associativity functional group in this annular oligopeptides can form in one or more rings them to connect, thereby makes this annular oligopeptides be constrained for single conformation, and epi-position formative amino acid in each region forms an epi-position.Can adopt many diverse ways to detect the single conformation that annular oligopeptides forms, this is known in the art.Can adopt the spectroscopic method of standard, as ¹h NMR spectrum, garden two chromatograms, rotating light dispersion or X-ray crystal pattern method are measured the conformation of this annular oligopeptides.

Epi-position formative amino acid forms an epi-position in each region, because annular oligopeptides is for this reason become single conformation by connection constraints in one or more rings.The epi-position energy specific binding target ligands forming.The ad hoc structure of epi-position and target ligands is unrestricted in the present invention.The object of this invention is to provide the oligopeptides support comprising by least two epi-position formative amino acid, its analogue or the appropriately combined multiple epi-positions that form of derivative, its array mode makes these epi-position formative amino acid can maintain the Stable conformation with enough rigidity so that epi-position can with specific target part (for example acceptor) mortise.Technician may know the amino acid whose sequence of epi-position formative, maybe can adopt for example method described in WO 01/01140 to prove and the interactional epi-position of target ligands.Technician do not need (knowing) the detailed chemical structure of epi-position likely because it is not so difficult to select to form the applicable amino acid of epi-position.In any given situation, the amino acid whose sequence of any epi-position formative may all be applicable to, and this depends on target ligands.In addition, the amino acid whose order in each region may determine the activity of the epi-position that forms, and this also depends on target ligands.Do not need to know the precise chemical structure structure of part, as long as know the biologic activity of target ligands.The epi-position formative amino acid being included in the present invention annular oligopeptides need not be known the precise chemical structure structure of its part, because can detect, the biology combined function of itself and part is measured epi-position and whether part interaction has occurred.According to the experimental data obtaining in corresponding detection model, technician is not difficult to select that this annular oligopeptides will comprise as the amino acid whose amino acid of epi-position formative, and has or not required activity to determine in each region, amino acid whose which kind of order can provide best biologic activity by mensuration.

Detect interactional test example attached bag between this annular oligopeptides and target ligands and draw together in conjunction with test, as used ELISA principle to detect those tests of combination between antibody and antigen.Other applicable in vitro tests comprises: the fluorescence of environment sensitive film combined with fluorescent probe changes, precipitin reaction, enhancer or inhibitor of enzymic activity etc.Can the test that also may be applicable to have the test that depends on tester and change cultured cell in vitro behavior, for example, in necrocytosis, cell proliferation, apoptosis, inhibition or activated cell Contact, secrete cytokines or other soluble product, synthetic specificity m-RNA, born of the same parents vesicle transhipment, changes cell signalling test etc.Also can carry out the in vivo test of animal or human's body, for example, in this annular oligopeptides, mix radio-labeled thing, then through various its subsequent distribution of administration " Invest, Then Investigate ".

In each region, form an epi-position.Therefore the epi-position number, forming in this annular oligopeptides is at least two, preferably two or three.Epi-position in this annular oligopeptides can be identical or different.In the time that the epi-position in this annular oligopeptides is not identical, different amino acid can be contained in each region, or contains amino acid identical but that order is different.An example of this epi-position is the epi-position being combined to form by Serine (S), phenylalanine (F) and arginine (R), and it can be in conjunction with the surface receptor of scavenger cell.The combination of epi-position and cell surface receptor can cause the inhibition of TNF secretion.

in ring, connect

The effect connecting in one or more rings is that this annular oligopeptides is constrained to the single conformation with multiple amino acid regions.Epi-position formative amino acid in each region is subject to tight constraint, and freedom of motion is very limited.In each like this region, just formed a stable epi-position, the stability that can improve and specificity produce biological answer-reply with target ligands interaction and react.

In each annular oligopeptides, the selection of associativity functional group and type thereof will be depended on epi-position formative amino acid and target ligands.Associativity functional group number and character may affect the biologic activity of this annular oligopeptides.Technician, by detecting whether there is required activity, is not difficult to select the associativity functional group of the most effective number or type.

Two or more associativity functional groups of suitably locating can form in one or more rings and connect.Preferably at least one associativity functional group is between each amino acid region.

In described one or more ring, connection can be covalently or non-covalently to connect.In this annular oligopeptides, associativity functional group can be identical or different.

This associativity functional group can be positioned on associativity amino acid.In one embodiment, this associativity functional group is the side chain of the amino acid whose side chain of described associativity or its modification or is added to the group on this associativity amino acid side chain.Following structural formula I is an example of this embodiment.

Or associativity functional group is arranged on the nitrogen-atoms of this annular oligopeptides ring peptide bond by replacing the hydrogen atom of being combined with nitrogen.Following formula II is an example of this embodiment.

In another embodiment, associativity functional group can be arranged on non-amino acid whose other the applicable group of oligopeptides ring.An example of this group is αhydroxycarboxylicacid, and its hydroxyl participates in fat key and non-peptide bond.

Phrase " two or more associativity functional groups " comprises following embodiment:

In the time that in ring, connection is non covalent bond,

This associativity functional group of ο is two side-chain radicals that are positioned at the oligopeptides ring on ring associativity amino acid, and they combine and form non-covalent connection;

This associativity functional group of ο is two side-chain radicals that are positioned at the oligopeptides ring on ring peptide bond nitrogen-atoms, and they combine and form non-covalent connection;

This associativity functional group of ο is two side-chain radicals that are positioned at the oligopeptides ring on other suitable groups of ring, and they combine and form non-covalent connection; Or

In the time that in ring, connection is covalent linkage,

This associativity functional group of ο is two groups that are positioned on ring associativity amino acid, and they combine, direct or form covalent attachment by joint as bifunctional group;

This associativity functional group of ο is two groups that are positioned on ring peptide bond nitrogen-atoms, and they combine, direct or form covalent attachment by joint as bifunctional group;

This associativity functional group of ο is positioned at two groups of ring on other suitable group, and they combine, direct or form covalent attachment by joint as bifunctional group.

Phrase " be positioned at ... on " refer to the direct combination of described conjugated group, or be connected on associativity amino acid, nitrogen-atoms or other suitable group by suitable joint.

In a preferred embodiment, in the one or more rings between associativity functional group, connection right and wrong are covalently bound.The present invention particularly preferably connects in non-covalent ring, keeps correct balance because can make like this between the rigidity and tenderness of this annular oligopeptides.The adequate rigidity of this annular oligopeptides so that peptide backbone remain on single conformation and guarantee the amino acid whose position of epi-position formative enough near and correct form can specific binding target ligands epi-position.The flexibility of this annular oligopeptides be also enough to allow the amino acid whose side chain sufficient movement of epi-position formative and adaptive its will in conjunction with the precision architecture of target ligands.Therefore, thus comprising stability that the present invention's annular oligopeptides of connecting in non-covalent ring can improve and specificity and the target ligands generation biological answer-reply that interacts reacts.

In described ring, connecting preferred hydrophobicity connects.In a particularly preferred embodiment, there are two amino acid regions and two lipotropy associativity functional groups.

Adopt in non-covalent ring and connect, particularly hydrophobic interaction has advantage, connects because the interaction between three or more associativity functional groups can form in ring.This will allow to form three or more amino acid regions, and this is to adopt when covalency ring is interior to be connected to be difficult to realize.Therefore, in other preferred implementation of the present invention, this annular oligopeptides can comprise three or more associativity functional groups, and they can form connection in ring and produce three or more amino acid regions.In the time that this annular oligopeptides is macromole, particularly preferably adopt lipotropy associativity functional group to form in non-covalent ring and connect by hydrophobic interaction.

Behind location, can form any molecule on any group that is positioned at suitable stereochemical structure that the associativity functional group connecting in one or more non-covalent rings can comprise the part that can form this annular oligopeptides ring.This associativity functional group is preferably placed on associativity amino acid or its analogue, and example is as described below, although also can adopt other group that can insert this annular oligopeptides ring.On general preferred described group, associativity functional group is connected in ring by peptide bond.In another embodiment, the connecting key of available other type substitutes one or more peptide bonds.The example of this connecting key is fat key, ehter bond, thioester bond and thioether bond.For the attack of limit protein enzyme in biological liquid, this may need sometimes.

Described associativity amino acid can comprise the natural amino acid with combined function group.

The preferred lipid amino acid analogue of described associativity amino acid.For example, associativity amino acid can comprise; Halfcystine, glycine, Methionin, aspartic acid or L-glutamic acid.Take halfcystine as example, described associativity functional group is added to the aliphatic group in cysteine side chain by sulfydryl functional group.Also can utilize the method to be added on Methionin, aspartic acid or L-glutamic acid side chain in connection with property functional group.Associativity amino acid or can be its to form side chain residue of associativity functional group be the amino acid of single aliphatic group.For example, if associativity amino acid take glycine as basis, the α hydrogen that forms this glycine of associativity functional group can be replaced by aliphatic group.

The above-mentioned aliphatic group that forms associativity functional group preferably preferably has 8-20 carbon atom, more preferably comprise 10-16 carbon atom, the most preferably aliphatic hydrocarbon chain of 10 or 12 carbon atoms, can be saturated or unsaturated straight chain or branched chain, unsubstituted or be substituted whole or in part (for example, by halogen atom).Or aliphatic group can be made up of silane component.

In following demonstrative structure I, described associativity amino acid is glycine, and its α hydrogen is by C ₁₀hydrocarbon chain replaces.

In one embodiment, described associativity functional group is positioned on the nitrogen-atoms of ring peptide bond.By with this associativity functional group, routine aliphatic group described above replaces the hydrogen atom on peptide bond nitrogen, can produce in non-covalent ring and connect, as hydrophobicity connects.

Can first use with suitable functional groups, as-SH ,-OH or-NH ₂methylene radical replace this hydrogen atom, then carry out derivatize with associativity functional group aliphatic group as previously discussed, thereby replace the hydrogen atom on the present invention annular oligopeptides peptide bond nitrogen with associativity functional group.

An aspect of this embodiment, the nitrogen-atoms of peptide bond has connected two epi-position formative amino acid in different aminoacids region, and annular oligopeptides of the present invention has following formula II:

In this formula, A, B, C, X, Y and Z represent epi-position formative amino acid; N represents the nitrogen-atoms of peptide bond between B/Z and C/Y, and Φ represents that the associativity functional group of nitrogen-atoms connection is therewith as above-mentioned aliphatic group.

Adopt non covalent bond, particularly another advantage of hydrophobic associativity functional group is to produce intramolecular interaction, and now the hydrophobicity associativity functional group of different annular oligopeptides interconnects and produces the dimer or the oligomer that contain along multiple repetition epi-positions of different directions orientation.This interactional degree depends on the sequence area of this annular oligopeptides, and it has determined the relative wetting ability of amino acid region.This can, by the wisdom of associativity functional group being selected to do further control, can change its chirality and volume size and accurately control the hydrophobic surface area that this oligopeptides exposes.Its advantage is intramolecular interaction adjustable and other internal constraint annular oligopeptides, or with other molecule as the interaction of protein or cyclodextrin.Its advantage is to produce the little polymer structure that comprises two or more the present invention's annular oligopeptides, this structure can be simultaneously in conjunction with two or more cell surface receptor, strongly start the trigger effect to cell thereby make by this way these acceptors be cross-linked, the reaction of priming signal transductory cascade.

The present invention's annular oligopeptides with other not identical interactional advantage of annular oligopeptides of the present invention is, because the different epi-positions of different oligopeptides can produce the polymer structure with several functions to the effect of this structure.For example, a kind of oligopeptides can comprise one or more epi-positions, after bind receptor, cause this polymer structure internalization, and after internalization, the second oligopeptides in this polymer structure can comprise and one or more epi-positions of the component interaction of intracellular signal transduction cascade reaction.

The interactional degree between the hydrophobic part that helps reduce different oligopeptides that is combined with of cyclodextrin and the present invention's annular oligopeptides, thereby prevent from forming large aggregate, because sterically hindered its activity that causes of epi-position reduces with respect to monomer or oligomer in these large aggregates.

Protein as albumin or gelatin solution in, in peptide the lipophilic portion of hydrophobicity associativity functional group with there is the protein joint area of alkyl or acyl group hydrocarbon chain avidity, cause one or more the present invention's annular oligopeptides to be attached to the surface of protein.This combination on one or more the present invention's annular oligopeptides and larger protein surface is a kind of mode of resetting, the so farthest triggering signal transduction of multiple epi-position arrays on this annular oligopeptides.

In another embodiment, it is covalently bound in the one or more rings between associativity functional group, connecting.In this kind of embodiment, associativity functional group also can be positioned on associativity amino acid.For example, associativity functional group can be the side chain of halfcystine (associativity amino acid), because disulfide linkage in the ring between these side chains has formed this connection.Or associativity amino acid can be Methionin and L-glutamic acid, they can be covalently bound by its side chain terminal group (associativity functional group) formation peptide bond, can be maybe can react to each other to form L-glutamic acid and the Serine of ester bond.Also can utilize these the amino acid whose analogues with same functional group.

In one embodiment, utilizable energy forms the hydrogen on peptide bond nitrogen-atoms in covalently bound associativity functional group substituted ring and realizes in ring covalently bound.This group can be thiol group, or end can form other covalently bound chain with functional group.Comprise with the hydrogen that associativity functional group replaces on the present invention annular oligopeptides peptide bond nitrogen-atoms, first use with suitable functional group as-SH ,-OH or-NH ₂methylene radical replace hydrogen atom, and then connect this functional group by second step with bifunctional reagent.This covalently bound formation comprises, use again with second methylene radical of suitable functional group and replace (being arranged on this annular oligopeptides appropriate position peptide bond nitrogen-atoms) second hydrogen atom, and in addition above-mentioned bifunctional reagent is connected to the functional group of the second methylene radical and between two nitrogen-atoms of this annular oligopeptides peptide bond, produces covalently bound.According to the character of mixing the associativity functional group in this annular oligopeptides, suitable bifunctional reagent can be those reagent that contain short chain, and each end of described short chain has and is selected from but is not limited to following listed functional group: carboxyl, amino, hydroxyl, sulfydryl, bromine, iodine, cyano group, azo-group and boronate.

In a particular aspects of the present invention, this annular oligopeptides comprises two amino acid regions and two associativity functional groups, be positioned at separately on associativity amino acid, in each region, three epi-position formative amino acid form an epi-position, and this annular oligopeptides can have following structural formula:

In this formula, A, B, C, X, Y and Z represent epi-position formative amino acid; A and X represent D amino acid, and B, C, Y and Z represent L amino acid; Or A and X represent L amino acid, B, C, Y and Z represent D amino acid; An epi-position is formed by C-A-B district, and an epi-position is formed by Z-X-Y district; Each ∑ represents the associativity amino acid with associativity functional group.

In an embodiment of the present invention's annular oligopeptides, Z-X-Y district and/or C-A-B district can be selected from aminoacid sequence RFS, RSF, FSR, FRS, SRF, SFR, QLS, QSL, SQL, SLQ, LQS or LSQ, and each ∑ is to contain C ₁₀straight chain hydrocarbon side chain is as the lipid amino acid of associativity functional group.Preferably Z-X-Y region is RFS, and in this region, preferably phenylalanine is D-type, and Serine is L-type, and arginine is L-type, and each lipid amino acid is L-type.One preferred embodiment in, Z-X-Y region and/or C-A-B region are selected from the sequence that above-mentioned each self-forming can suppress the epi-position of TNF secretion.Therefore, it is the disease that worsens the factor that these sequences can be used for treating TNF, for example, be selected from the disease of cancer, obesity, heart disease, autoimmune disease and inflammation.Can treat the autoimmune disease that is selected from rheumatoid arthritis, multiple sclerosis and Crohn's disease by these sequences.

In said structure, C-A-B district can be identical or different with Z-X-Y district.

In another aspect of this invention, this annular oligopeptides comprises three amino acid regions, in each region, forms an epi-position by three epi-position formative amino acid, and preferably this oligopeptides comprises three associativity functional groups.

In another aspect of this invention, this annular oligopeptides comprises Two Areas, in each region, forms an epi-position by 4 epi-position formative amino acid.In the present invention's a kind of embodiment in this respect, this annular oligopeptides has following structural formula:

In this formula, A, B, C, D, W, X, Y and Z represent epi-position formative amino acid; An epi-position is formed by D-C-A-B district, and an epi-position is formed by Z-X-Y-W district; Each ∑ represents an associativity amino acid.

In the present invention's another kind of embodiment in this respect, this annular oligopeptides has following structural formula:

In this formula, A, B, C, D, W, X, Y and Z represent epi-position formative amino acid; An epi-position is formed by D-C-A-B district, and an epi-position is formed by Z-X-Y-W district; Each N represents the nitrogen-atoms of peptide bond between B-Z and C-Y, and each Φ represents an associativity functional group being connected with this nitrogen-atoms.

In the present invention in this respect in an embodiment of annular oligopeptides, Z-X-Y-W district and/or D-C-A-B district are selected from: aminoacid sequence YEKA, YEAK, YAKE, YAEK, YKAE, YKEA, EYKA, EYAK, EKAY, EKYA, EAKY, EAYK, KAYE, KAEY, KYAE, KYEA, KEAY, KEYA, AKEY, AKYE, AYEK, AYKE, AEYK or AEKY, each ∑ is that the straight chain hydrocarbon side chain of the hydrocarbon chain that comprises a long 8-20 carbon atom is in conjunction with the aliphatic amino acid of functional group.Preferred Z-X-Y-W district is YEKA, and D-C-A-B district is EYAK.In a preferred embodiment, Z-X-Y-W district and/or D-C-A-B district are selected from the sequence of the epi-position of above-mentioned each self-forming energy protease inhibition hydrolytic activity.Therefore, it is the disease that worsens the factor that these sequences can be used for treating protease activity, as cardiovascular disorder, blood circulation disease and HIV.

In the present invention's a kind of embodiment in this respect, D-C-A-B district and Z-X-Y-W district can be identical.

composition

On the other hand, the invention provides the pharmaceutical composition that comprises annular oligopeptides defined above and pharmaceutically acceptable vehicle and/or co-adjuvant.Described vehicle is selected from: diethylene glycol monoethyl ether (transcutol), poloxamer segmented copolymer, cyclodextrin, nonionogenic tenside or cholate.If described vehicle is nonionogenic tenside, be preferably selected from the acyl ester of polyoxyethylene glycol or the aliphatic ether of polyoxyethylene glycol.

As previously discussed, in one embodiment of the invention, can between different annular oligopeptides, form in ring and connect, now the hydrophobicity associativity functional group of different annular oligopeptides is connected to each other and produces dimer or the oligomer of the multiple repetition epi-positions that contain different orientation.Can jointly mix to regulate the degree reacting to each other in this ring with the vehicle that can help hydrophobic molecule to be dissolved in aqueous medium.

application

On the other hand, the invention provides the application as medicine, prevention or diagnostic preparation of annular oligopeptides defined above or composition defined above.

It is the application that worsens the medicine of factor disease for the manufacture for the treatment of TNF that the present invention also provides annular oligopeptides defined above or composition defined above.This class disease is preferably selected from: obesity, heart disease, autoimmune disease and inflammatory disease.More preferably this class disease is rheumatoid arthritis or Crohn's disease.Available annular oligopeptides treatment TNF defined above is the cancer that worsens the factor.This cancer can be optimum or pernicious.Preferably solid tumor of this cancer.This cancer includes but not limited to: ovarian cancer, mammary cancer, skin carcinoma and epithelial cancer.

It is the disease that worsens the factor for the manufacture for the treatment of protease activity that the present invention also provides annular oligopeptides defined above or composition defined above, comprises the application of the medicine of cardiovascular disorder, blood circulation disease and HIV.In a specific implementations of the present invention, each district inclusion epi-position formative amino acid SRER, SERE, EYKA, YEAK, SFR or RFS in this annular oligopeptides, as described in Example 7, the proteolytic activity of their energy Trombin inhibitings.

In a specific implementations of the invention described above, this annular oligopeptides comprises Serine, phenylalanine and arginine as epi-position formative amino acid at least one region.Prove that these oligopeptides have the activity that suppresses scavenger cell TNF secretion.This embodiment one preferably aspect, described associativity functional group is with C ₁₂the lipid amino acid of hydrocarbon side chain, epi-position formative amino acid is selected from RFS, FSR or SRF, and its central amino acid is D-type, and two, outside amino acid is L-type.Each epi-position in this annular oligopeptides can be identical or different.

Because these oligopeptides are lowered the ability of TNF secretion, they can effectively be treated such as rheumatoid arthritis, Crohn's disease, multiple sclerosis, obesity, heart disease, autoimmune disease and relate to the Other diseases of inflammatory process.Because these oligopeptides are lowered the ability of TNF secretion, they also can effectively treat TNF is the cancer that worsens the factor.This cancer can be optimum or pernicious.Preferably solid tumor of this cancer.This cancer includes but not limited to: ovarian cancer, mammary cancer, skin carcinoma and epithelial cancer.

In a specific implementations of the invention described above, this annular oligopeptides comprises amino acid A, K, E and Y as epi-position formative amino acid in its at least one region.Prove that these oligopeptides have the activity that suppresses scavenger cell TNF secretion.This embodiment one preferably aspect, described associativity functional group is with C ₁₀the lipid amino acid of hydrocarbon side chain, epi-position formative amino acid is selected from YEKA or EYAK.Each epi-position in this annular oligopeptides can be identical or different.One contains amino acid A, K, the random copolymers of E and Y is examined and is sent ketone (Copaxone) (acetic acid lattice draw the ability of lowering the TNF secretion in body by this compound for the effect of thunder (Glatiramer acetate) treatment multiple sclerosis to reflect (referring to Weber MS, Starck M, Wagenpfeil S, Meinl E, Hohlfeld R and Farina C. " multiple sclerosis: acetic acid lattice draw for thunder inside and outside and suppress monocytic reactivity " (Multiplesclerosis:glatiramer acetate inhibits monocyte reactivity in vitro and invivo) Brain 127 1370-8 pages (2004)).Put at this point, multiple sclerosis is treated in the annular oligopeptides cyclisation (A-K-∑-Y-E-K-A-∑-E-Y) that also can detect with embodiment 6, because the same amino acid that it contains nonrandom configuration, has same function to scavenger cell TNF secretion.Also can treat multiple sclerosis with A, K, E and amino acid whose other collating sequence of Y in the present invention's annular oligopeptides.

An advantage of the invention is, compared with conventional biological receptor, can obtain high specificity binding interactions with annular oligopeptides of the present invention.This annular oligopeptides is less, preferably contains and is no more than 6 or 8 amino acid.Therefore, the immunogenicity protein corresponding with it of the present invention of preparation annular oligopeptides is compared much smaller.

Content according to an aspect of the present invention, by annular oligopeptides of the present invention be mixed with not only in vitro can with ligand interaction, and this composition also can body in application.

Can be by any approach adapting with disease to be treated, include but not limited to oral, intranasal, per rectum, per os cheek, hypogloeeis, through lung, vagina, part, through eyes, in ear, subcutaneous, intracutaneous, intraarticular, sheath, in intramuscular, brain, encephalic and intravenous administration approach, give annular oligopeptides of the present invention.

Can free-water solution form, or combine with the drug excipient in the present composition and give this annular oligopeptides.Also this annular oligopeptides or composition and other can be had to synergy, after maybe strengthening that its active bioactive molecule is common and mixing, as medicine.When by certain approach, during as oral or per rectum administration, this oligopeptides or composition can be mixed with to solid, semisolid or liquid, be filled in capsule, or as solid compacting in flakes, or be pressed into ball.Now, if final route of administration is oral, described capsule, tablet or pill can have enteric coating.Gel, paste or finish are particularly suitable for topical application, this oligopeptides can adopt aerosol form through lung or nasal administration.

method

On the other hand, the invention provides the method for preparation annular oligopeptides defined above, comprising:

I) select epi-position formative amino acid;

Ii) the amino acid whose annular oligopeptides of this epi-position formative has been mixed in preparation.

The method of identifying epi-position is known.In traditional combinational chemistry, must synthesize hundreds of different groups (as amino acid) with different order composition may combine and detect efficiency separately, just can identify the most promising sequence in conjunction with certain specific receptors.This kind method is consuming time, expense is high, be subject to the restriction of the adopted chemical reaction character that heterogeneity is linked together.

As mentioned above, epi-position formative amino acid can be selected by many diverse ways well known by persons skilled in the art.

WO 01/01140 provide a kind of method measure formation can with the molecule of the epi-position of required ligand interaction, the method adopts the multiple different conjugates that are assembled into by non covalent bond.As described in WO01/01140 2-5 page, each conjugate contains a base and tail base.The tail base of conjugate forms hydrophobicity aggregate, each conjugate each other can free movement in assemblage, therefore in the time there is part, at least two stature bases (identical or different) by appropriate location form can with the epi-position of ligand interaction, its interaction strength is higher than a base independent situation separately.Base is conventionally hydrophilic, and tail base is conventionally hydrophobic, the lipophilic group being for example made up of hydrocarbon chain, halophile fluorocarbon chain structure or silylation.Build the conjugate containing a base and tail base, its tail base can, in conjunction with forming hydrophobicity aggregate, generally form supramolecule assemblage, and as micella, laminated structure, liposome or other lipid conformation, it is closely close that in water, the orientation of this conjugate makes each base.Because this conjugate is movable in assemblage, therefore its base can be taked many different positions in assemblage.Therefore conventionally not identical base can free movement in assemblage, surprisingly, and their induction biological results that can act synergistically, and former each the base oneself before this of this result can not be induced.

In a preferred implementation of the present invention, adopt the disclosed method of WO 01/01140 to select epi-position formative amino acid, this method comprises:

(a) select one group of conjugate, each conjugate comprises a base and tail base, and a basic matrix row, and wherein every stature base comprises amino acid;

(b) form non-covalent connection at them, wherein tail base hydrophobicity assemble and this conjugate is movably;

(c) detect between non-covalent connection and target ligands and interact fully;

(d) one group of conjugate repeating step (a) of an optional basic matrix row with containing modification-(c); With

(e) once find to interact fully in step (c), select the amino acid of this group conjugate head base of composition as the epi-position formative amino acid of step (a).

The example that detects " whether fully interacting " test comprises in conjunction with test, for example, utilize ELISA principle to detect those tests of combination between antibody and antigen.Other applicable in vitro tests comprises the change in fluorescence of the membrane-bound fluorescent probe of environment sensitive, precipitin reaction, enhancer or inhibitor test of enzymic activity etc.Can the test that also may be applicable to have the test that depends on tester and change cultured cell in vitro behavior, for example, in necrocytosis, cell proliferation, apoptosis, inhibition or activated cell Contact, secrete cytokines or other soluble product, synthetic specificity m-RNA, born of the same parents vesicle transhipment, changes cell signalling test etc.Also can carry out the in vivo test of animal or human's body, for example, in this supramolecule assemblage, mix radio-labeling, then through various its subsequent distribution of administration " Invest, Then Investigate ".

Root accordingly, is prepared a series of different supramolecules assembling matter (or " probe ") by combined method, contains separately the various combination that is selected from pre-synthesis conjugate storehouse.Can select suitable conjugate according to the known properties of target ligands, can utilize simply the unusual base of broad range to improve two or more bases to form the probability of this kind of part epi-position.In this way, whether fully detect subsequently interaction between above-mentioned probe and part, can be by further increasing a base, remove some base, or these two kinds of modes modify, whether the interaction that again detects synthetic probe is abundant, to find most promising conjugate combination.Finally, identify and select most promising base combination as epi-position formative amino acid.

Embodiment

Embodiment 1: it is the dose response of TNF secretion that annular oligopeptides suppresses lipopolysaccharides (LPS) stimulating expression of macrophage

1. synthesize and there is structural formula with the standard method of Merrifield resin base technology: the annular octapeptide of annular (DF-S-∑-R-DF-S-∑-R-), then cyclisation in solution phase.Prepare the solution of this purified peptide with distilled water, concentration 1mg/ml.

R, F and S represent epi-position formative amino acid, and ∑ representative is positioned at the associativity functional group on associativity amino acid, and wherein associativity amino acid is L-α aminocarboxylic acid, and associativity functional group is the side chain residue containing the aliphatic straight chain of 10 carbon atoms.

All amino acid is all L-type, except as otherwise noted.

All amino acid is connected with peptide bond.

24 hole colony plates institute porose middle inoculation J774A.1 cell (a kind of macrophage system), the every hole 5x10 of inoculum density ⁵individual cell/ml (every hole 1ml RPMI1640 nutrient solution), at 37 ℃ and 5%CO ₂overnight incubation in/air.

3. the octapeptide solution of step 1 was joined in 15 holes that step 2 contains cell in second day to ultimate density 50,25,12.5,6.25 or each three holes of 3.125ug/ml.All the other hole indwellings do not deal with.Cultivate again these dull and stereotyped 4 hours for 37 ℃.

4. add lipopolysaccharides (deriving from e. coli strains 0111B4) in three holes accepting the hole of this octapeptide solution and do not accept peptide solution.The final concentration of lipopolysaccharides is 0.625ug/ml.

5. flat board is cultivated and spent the night, inferior daily commercialization ELISA test kit detects the TNF of supernatant liquor.

Following table shows the result obtaining, and shows that this annular oligopeptides suppresses the activity of the TNF secretion that LPS stimulates relevant to dosage.

Peptide concentration (ug/ml) TNF concentration (pg/ml) standard deviation

0 1780.09 136.49

3.125 1494.53 127.83

6.25 1346.20 45.84

12.5 1037.31 90.01

25.0 621.75 14.53

50.0 613.70 15.46

Embodiment 2: suppressing cholera toxin B (CTB) fragment stimulating expression of macrophage containing the annular oligopeptides of different associativity functional group is the effect of TNF secretion

1. preparation has the annular oligopeptides of following structural formula:

Annular (DF-R-∑ ₁₀-S-DF-R-∑ ₁₀-S-)

Annular (DF-S-∑ ₁₀-R-DF-S-∑ ₁₀-R-)

Annular (DF-R-∑ ₄-S-DF-R-∑ ₄-S-)

Annular (DF-S-∑ ₄-R-DF-S-∑ ₄-R-)

In this formula, S, F and R represent epi-position formative amino acid, and ∑ representative is positioned at the associativity functional group on associativity amino acid, ∑ ₁₀represent racemize associativity amino acid, it is a kind of aminocarboxylic acid, and associativity functional group is by branchiess-C ₁₀h ₂₁the side chain residue forming, ∑ ₄represent nor-leucine, the associativity functional group in formula is 4 carbon side chains.

With diethylene glycol monoethyl ether (transcutol), these annular oligopeptides are made into the solution of concentration 5mg/ml, then with distilled water diluting to concentration 1mg/ml.

24 hole colony plates institute porose middle inoculation J774A.1 cell (a kind of macrophage system), the every hole 3x10 of inoculum density ⁵individual cell/ml (every hole 1ml RPMI1640 nutrient solution), at 37 ℃ and 5%CO ₂overnight incubation under/air.

3. the octapeptide solution of step 1 was joined in 12 holes that step 2 contains cell in second day to each three holes of ultimate density 12.5ug/ml.All the other hole indwellings do not deal with.Cultivate again these dull and stereotyped 4 hours for 37 ℃.

4. accepting the hole of this octapeptide solution and not accepting to add cholera toxin B (CTB) fragment in three holes of peptide solution.The concentration of cholera toxin B (CTB) fragment is 10ug/ml.

Following table shows the result obtaining, and shows to exist without branch-C ₁₀h ₂₁the long chain hydrocarbon side chain of composition, as associativity functional group, can cause this annular oligopeptides to produce the activity that suppresses cholera toxin B (CTB) fragment stimulation TNF.And the annular oligopeptides of the associativity functional group of containing 4 carbon chain length does not have any inhibition activity.

Embodiment 3: suppressing lipopolysaccharides (LPS) stimulating expression of macrophage containing the annular oligopeptides of different epi-position formative amino acid and associativity amino acid three-dimensional chemical configuration is the effect of TNF secretion

1. preparation has the annular octapeptide of following structural formula:

Annular (DF-S-∑ _l-R-DF-S-∑ _l-R-)

Annular (DF-S-∑ _l-R-DF-S-∑ _d-R-)

Annular (DF-S-∑ _d-R-DF-S-∑ _d-R-)

Annular (DF-R-∑ _l-S-DF-R-∑ _l-S-)

Annular (DF-R-∑ _l-S-DF-R-∑ _d-S-)

Annular (DF-R-∑ _d-S-DF-R-∑ _d-S-)

In this formula, F, S and R are epi-position formative amino acid, ∑ _lrepresent position associativity amino acid, it is a kind of L-α aminocarboxylic acid, and wherein associativity functional group is by without branch-C ₁₀h ₂₁the side chain residue forming; ∑ _drepresent associativity amino acid, it is D-α aminocarboxylic acid, and wherein associativity functional group is by without branch-C ₁₀h ₂₁the side chain residue forming.These annular oligopeptides are made into concentration 1mg/ml with distilled water.

3. the octapeptide solution of step 1 was joined in 18 holes that step 2 contains cell in second day to each three holes of ultimate density 50ug/ml.All the other hole indwellings do not deal with.Cultivate again these dull and stereotyped 4 hours for 37 ℃.

4. accepting the hole of this octapeptide solution and not accepting to add lipopolysaccharides (deriving from e. coli strains 0111B4) in three holes of peptide solution.The final concentration of lipopolysaccharides is 1.25ug/ml.

Following table shows the result obtaining, and showing to contain the amino acid whose annular oligopeptides inhibition LPS of order R-DF-S stimulates the activity of TNF secretion very high, and requiring at least one associativity amino acid is that L-α aminocarboxylic acid, its associativity functional group are without branch-C ₁₀h ₂₁the side chain residue forming, another associativity amino acid is the identical aminocarboxylic acid of L-type or D-type.

Cyclodextrin and being combined with of oligopeptides of the present invention's annular help reduce interactional level between different oligopeptides hydrophobic parts, thereby prevent from forming large aggregate, due to the epi-position Existential Space steric hindrance in these large aggregates, cause specific activity monomer or the oligomer of this aggregate low.

Embodiment 4: with the hydroxypropylβ-cyclodextrin of wide concentration range with contain epi-position formative amino acid R- _dthe annular oligopeptides of interior constraint of the epi-position that F-S forms, suppressing lipopolysaccharides (LPS) stimulating expression of macrophage is the dosage relevant effect of TNF secretion

With diethylene glycol monoethyl ether (transcutol) will have structural formula cyclisation ( _df-S-∑-R- _df-S-∑-R-) annular octapeptide be made into the solution of concentration 5mg/ml, in formula, the associativity amino acid of ∑ representative is L-α aminocarboxylic acid, the associativity functional group being positioned on associativity amino acid is the side chain residue forming containing the aliphatic straight chain of 10 carbon atoms.

2. the solution of the 200ul step 1 of 6 independent equal portions is transferred in new 8ml test tube, slowly vibration be respectively by the concentration that distilled water is prepared 50,25,12.5,6.25,3.125 or the 800ul hydroxypropylβ-cyclodextrin of 1.5625mg/ml mix.

24 hole colony plates institute porose middle inoculation J774A.1 cell (a kind of macrophage system), the every hole 5x10 of inoculum density ⁵individual cell/ml (every hole 1ml RPMI1640 nutrient solution), at 37 ℃ and 5%CO ₂overnight incubation under/air.

3. each solution of step 1 was joined in the hole that step 2 contains cell in second day to each three holes of ultimate density 8,4 or 2ug/ml with proper volume.All the other hole indwellings do not deal with.Cultivate again these dull and stereotyped 4 hours for 37 ℃.

4. accepting the hole of this octapeptide solution and not accepting to add lipopolysaccharides (deriving from e. coli strains 0111B4) in three holes of peptide solution.The final concentration of lipopolysaccharides is 0.625ug/ml.

Following table shows the result obtaining, and shows that this octapeptide suppresses LPS and stimulates the activity of TNF secretion relevant to dosage.

Peptide concentration cyclodextrin and peptide TNF concentration

Standard deviation

(ug/ml) weight ratio (pg/ml)

0 - 2519.3 64.4

2 40 2450.2 61.2

2 20 2223.9 37.4

2 10 2278.3 89.4

2 5 2177.2 110.9

2 2.5 2314.2 101.8

2 1.25 2161.8 94.7

4 40 1646.5 98.4

4 20 1488.1 16.6

4 10 1474.8 80.6

4 5 1477.5 33.2

4 2.5 1538.7 80.4

4 1.25 1599.9 106.4

8 40 1276.9 106.5

8 20 1298.4 17.3

8 10 1218.7 20.6

8 5 1140.6 34.5

8 2.5 1105.7 35.9

8 1.25 1130.9 71.8

Result shows, has significantly suppressed TNF secretion containing the octapeptide preparation of cyclodextrin, in tissue culture hole the density loss of TNF to 2ug/ml, extensively 40: 1 wt of concentration range: the cyclodextrin of wt has been realized this inhibition.

Embodiment 5: contain epi-position formative amino acid R-with hydroxypropylβ-cyclodextrin preparation _dthe annular oligopeptides of interior constraint of the epi-position that F-S forms, the effect of inhibition lipopolysaccharides (LPS) stimulation in rats TNF secretion

1. as described in Example 4, with diethylene glycol monoethyl ether and hydroxypropylβ-cyclodextrin will have structural formula cyclisation ( _df-S-∑-R- _df-S-∑-R-) annular octapeptide wiring solution-forming, in this formula, the associativity amino acid of ∑ representative is L-α aminocarboxylic acid, the associativity functional group being positioned on this associativity amino acid is the side chain residue forming containing the aliphatic straight chain of 10 carbon atoms.Peptide solution concentration is 1mg/ml, and the final concentration of cyclodextrin is 40mg/ml.

2. give the rat i.p injection 1ml peptide solution (every rat 1mg peptide) of 250 grams of body weight.Second group of rat accepted diethylene glycol monoethyl ether/cyclodextrin vehicle that 1ml does not contain peptide.After one hour, then to rat injection 1mg lipopolysaccharides (Salmonella abortus equi).

After 3.150 minutes, get blood sample ELISA and detect the TNF level of two groups.The result that following table shows shows that this Toplink suppresses TNF generation in to the responsing reaction stimulating, and makes it to decline more than 75%.

Vehicle peptide

TNF(pg/ml) 10717 2370

SD 6635 1831

Every treated animal number 35

Embodiment 6: examine and send the annular analogue of ketone (acetic acid lattice draw for thunder) to suppress TNF secretion

1. preparation has the annular oligopeptides of following structural formula:

Annular (A-K-∑-Y-E-K-A-∑-E-Y-)

Annular (A-K-∑-E-Y-K-A-∑-Y-E-)

All amino acid is all L-type, except as otherwise noted.

All amino acid is connected with peptide bond.

A, K, Y and E represent epi-position formative amino acid, and ∑ representative is positioned at the associativity functional group on associativity amino acid, and described associativity amino acid is L-α aminocarboxylic acid, and associativity functional group is the side chain residue forming containing the aliphatic straight chain of 10 carbon atoms.

With hexafluoro-Virahol (HFIP), this annular oligopeptides is mixed with to the solution of concentration 10mg/ml, then mixes containing the HFIP solution of concentration 50mg/ml β-hydroxypropyl cyclodextrin with equal-volume.Under nitrogen, dry up this organic solution, peptide/cyclodextrin complexes is heavily dissolved in to distilled water, obtain the annular oligopeptides liquid of ultimate density 1mg/ml.

24 hole colony plates institute porose middle inoculation J774A.1 cell (a kind of macrophage system), the every hole 3x10 of inoculum density ⁵individual cell/ml (every hole 1ml RPMI RPMI-1640), at 37 ℃ and 5%CO ₂overnight incubation under/air.

3. the octapeptide solution of step 1 was joined in the hole that step 2 contains cell in second day to each three holes of ultimate density 12.5ug/ml.Also prepared other hole that only contains 62.5ug/ml cyclodextrin.All the other hole indwellings do not deal with.Cultivate again these dull and stereotyped 4 hours for 37 ℃.

4. accept the hole of this oligopeptide solution and do not accept to add lipopolysaccharides (deriving from e. coli strains 0111B4) in three holes of peptide solution.The final concentration of lipopolysaccharides is 0.1ug/ml.

This table shows the result obtaining, and shows that annular oligopeptides a kind of but not two kinds of tests have the effect that suppresses LPS stimulation TNF secretion.Therefore the aminoacid sequence in each region has affected the biologic activity of this oligopeptides.Annular (A-K-∑-Y-E-K-A-∑-E-Y) can be used for treating multiple sclerosis, because it contains and examines the amino acid (unless random configuration is outer) of sending ketone identical and show that scavenger cell TNF secretion is had to same function.

Embodiment 7: the activity of Trombin inhibiting

1. the synthetic annular oligopeptides with following structural formula as previously discussed, prepares the complex liquid of itself and cyclodextrin, concentration 1mg/ml with Chinese Gus (Hanks) balanced salt solution by the method for embodiment 6.

(i) annular (R-E-∑-S-R-E-R-∑-S-E-)

(ii) annular (A-K-∑-E-Y-K-A-∑-Y-E-)

(iii) annular (dF-R-C-S-dF-R-C-S-)

(iv) annular (dF-S-C-R-dF-S-C-R-)

All amino acid is all L-type, except as otherwise noted.

All amino acid is connected with peptide bond.

In formula, R, E, S, Y, K and A represent epi-position formative amino acid, ∑ representative is positioned at the associativity functional group on associativity amino acid, described associativity amino acid is L-α aminocarboxylic acid, associativity functional group is the side chain residue containing the aliphatic straight chain of 10 carbon atoms, C represents halfcystine, cross structure in the oxidized formation of halfcystine of each ring in formula.

2. 20ul thrombin solution (0.01mg/ml) is assigned in the hole of 394-hole droplet plate, mixes with 60ul annular oligopeptide solution.

3. each hole adds 20ul substrate boc-β-phenmethyl-Asp-Pro-Arg-7-amido-4-hydrochloric acid tonka bean camphor (0.01mg/ml), the time course that detection substrate produces.

4. the result showing from following table can be known, the proteolytic activity effect difference of the annular oligopeptides Trombin inhibiting of studying, and these active different amino acid character included with ring are relevant with combination.Can understand, by selecting properly amino acid, can produce enough structures to enzymic activity performance remarkable effect of rigidity according to affined annular oligopeptides template.

Therefore, it is the disease that worsens the factor that the annular oligopeptides that can adopt the present invention to have Trombin inhibiting proteolytic activity is treated protease activity, comprises cardiovascular disorder, blood circulation disease and HIV.

From then on table is visible, and according to the amino-acid sequence in each region, some annular oligopeptides has the activity stronger than other oligopeptides.

Embodiment 8: suppress TNF secretion containing the annular oligopeptides of two different epi-positions

1. preparation has two annular oligopeptides of following structural formula:

(i) annular (dF-S-∑-Q-dL-S-∑-R-)

(ii) annular (dF-S-∑-L-dQ-S-∑-R)

All amino acid is all L-type, except as otherwise noted.

All amino acid is connected with peptide bond.

In formula, Q, L, S, R and F represent epi-position formative amino acid, ∑ representative is positioned at the associativity functional group on associativity amino acid, described associativity amino acid is L-α aminocarboxylic acid, and associativity functional group is the side chain residue forming containing the aliphatic straight chain of 10 carbon atoms.

As described in Example 4, prepare the diethylene glycol monoethyl ether/cyclodextrin soln of these annular oligopeptides, the ultimate density of peptide is 1mg/ml, and cyclodextrin is 20mg/ml.

Three blocks of 24 hole colony plates institute porose middle inoculation J774A.1 cell (a kind of macrophage system), the every hole 3x10 of inoculum density ⁵individual cell/ml (every hole 1ml RPMI RPMI-1640), at 37 ℃ and 5%CO ₂overnight incubation under/air.

3. the peptide solution of step 1 was joined in the hole that step 2 contains cell in second day, annular oligopeptides ultimate density is 50,25,12.5,6.25 or each three holes of 3.125ug/ml.Also preparation is containing the additional bore of cyclodextrin 1,0.5,0.25,0.125 and 0.0625mg/ml.All the other hole indwellings do not deal with.Cultivate again these dull and stereotyped 4 hours for 37 ℃.

4. add lipopolysaccharides (deriving from e. coli strains 0111B4) in three holes accepting the hole of this octapeptide solution and do not accept peptide solution.The final concentration of lipopolysaccharides is 0.1ug/ml.

Following table shows the result obtaining, and shows containing two different epi-positions, and an annular oligopeptides being made up of F, R and S amino acid can be secreted with the TNF of dosage dependence form downward J774 cell.

^*reading is higher than detecting maximum limit

Claims

1. the affined annular oligopeptides in inside, it is characterized in that, the ring being formed by least 6 amino acid that described oligopeptides comprises energy specific binding target ligands, wherein said ring comprises multiple amino acid regions, at least two epi-position formative amino acid are contained in each region, and two or more associativity functional groups, the location of described associativity functional group forms in one or more non-covalent rings them to connect; Described annular oligopeptides is constrained for single conformation thus, makes the epi-position formative amino acid in each region form epi-position, each epi-position energy specific binding target ligands; Described associativity functional group respectively do for oneself saturated or unsaturated, the straight or branched of 8-20 carbon atom, the aliphatic hydrocarbon chain that do not replace or replace;

The structure of described annular oligopeptides is selected from:

In formula, A, B, C, X, Y and Z represent epi-position formative amino acid; A and X represent D amino acid and B, C, Y and Z represent L amino acid, or A and X represents L amino acid and B, C, Y and Z represent D amino acid; An epi-position is formed by C-A-B district, and an epi-position is formed by Z-X-Y district; Described Z-X-Y district and/or C-A-B district are selected from aminoacid sequence RFS, RSF, FSR, FRS, SRF, SFR, QLS, QSL, SQL, SLQ, LQS or LSQ, and each Σ is associativity amino acid and is lipid amino acid; With

In formula, A, B, C, D, W, X, Y and Z represent epi-position formative amino acid; An epi-position is formed by D-C-A-B district, and an epi-position is formed by Z-X-Y-W district; Described Z-X-Y-W district and/or D-C-A-B district are selected from aminoacid sequence YEKA, YEAK, YAKE, YAEK, YKAE, YKEA, EYKA, EYAK, EKAY, EKYA, EAKY, EAYK, KAYE, KAEY, KYAE, KYEA, KEAY, KEYA, AKEY, AKYE, AYEK, AYKE, AEYK or AEKY, and each Σ is associativity amino acid and is lipid amino acid.

2. annular oligopeptides as claimed in claim 1, is characterized in that, the regional in described oligopeptides comprises 3 epi-position formative amino acid, and these three epi-position formative amino acid have three-dimensional chemical configuration alternately; And/or described amino acid is amino acid, amino acid analogue or its D-type amino acid of natural origin.

3. annular oligopeptides as claimed in claim 1, is characterized in that, in the one or more rings between described associativity functional group, connecting is that hydrophobicity connects.

4. annular oligopeptides as claimed in claim 1, is characterized in that, at least one associativity functional group is between each region.

5. annular oligopeptides as claimed in claim 4, is characterized in that, each epi-position formative amino acid that adjoins associativity functional group has the identical three-dimensional chemical configuration of associativity functional group adjoining with this.

6. annular oligopeptides as claimed in claim 1, is characterized in that, described epi-position is identical.

7. the annular oligopeptides as described in any one in claim 3-6, is characterized in that, described associativity functional group is positioned on lipid amino acid, and described associativity functional group is C ₈-C ₂₀straight chain hydrocarbon side chain.

8. annular oligopeptides as claimed in claim 7, is characterized in that, described straight chain hydrocarbon side chain is C ₁₀-C ₁₆straight chain hydrocarbon side chain.

9. the annular oligopeptides as described in any one in claim 3-6, is characterized in that, described associativity amino acid is halfcystine, and described associativity functional group links the aliphatic C in cysteine side chain by sulfydryl ₈-C ₂₀hydrocarbon chain.

10. annular oligopeptides as claimed in claim 1, is characterized in that, described Z-X-Y district and/or C-A-B district form the epi-position that can suppress TNF secretion.

11. annular oligopeptides as claimed in claim 1, is characterized in that, described Z-X-Y district is RFS, and/or the each self-forming in described Z-X-Y district and C-A-B district can suppress the epi-position of TNF secretion.

12. annular oligopeptides as claimed in claim 11, is characterized in that, phenylalanine is D type, and Serine is L-type, and arginine is L configuration, and each aliphatic amino acid is L-type.

13. annular oligopeptides as claimed in claim 1, is characterized in that, described C-A-B district is identical with Z-X-Y district.

14. annular oligopeptides as claimed in claim 1, is characterized in that, described Z-X-Y-W district is YEKA, and described D-C-A-B district is EYAK, and/or the epi-position of described Z-X-Y-W district and D-C-A-B district each self-forming energy protease inhibition hydrolytic activity.

15. annular oligopeptides as claimed in claim 1, is characterized in that, described D-C-A-B district is identical with Z-X-Y-W district.

16. annular oligopeptides as claimed in claim 1, is characterized in that, described annular oligopeptides comprises Serine, phenylalanine and arginine as epi-position formative amino acid at least one region, and each epi-position forms the epi-position that can suppress TNF secretion.

17. annular oligopeptides as claimed in claim 1, as medicine, prevention or diagnostic preparation.

It is the application worsening in the medicine of disease of the factor that 18. annular oligopeptides as claimed in claim 1 are used for the treatment of TNF in manufacture.

19. application as claimed in claim 18, is characterized in that, described disease is selected from cancer, obesity, heart disease, autoimmune disease and inflammatory disease.

20. application as claimed in claim 19, is characterized in that, described disease is the autoimmune disease that is selected from rheumatoid arthritis, multiple sclerosis or Crohn's disease.

It is the application worsening in the medicine of disease of the factor that 21. annular oligopeptides as claimed in claim 1 are used for the treatment of protease activity in manufacture, the epi-position of the Z-X-Y-W district of described annular oligopeptides and/or D-C-A-B district each self-forming energy protease inhibition hydrolytic activity.

22. application as claimed in claim 21, is characterized in that, described disease is selected from: cardiovascular disorder, circulatory diseases and HIV.

23. 1 kinds of pharmaceutical compositions, it comprises the annular oligopeptides described in any one and pharmaceutically acceptable vehicle and/or co-adjuvant in claim 1-17.

24. pharmaceutical compositions as claimed in claim 23, is characterized in that, described vehicle is selected from: diethylene glycol monoethyl ether, poloxamer segmented copolymer, cyclodextrin, nonionogenic tenside or cholate.

25. pharmaceutical compositions as claimed in claim 24, is characterized in that, described nonionogenic tenside is selected from the acyl ester of polyoxyethylene glycol or the aliphatic ether of polyoxyethylene glycol.

26. 1 kinds prepare can specific binding target ligands as the method for annular oligopeptides that any one defines in claim 1-17, described method comprises:

I) provide epi-position formative amino acid;

Ii) the amino acid whose annular oligopeptides of described epi-position formative is mixed in preparation.

27. methods as claimed in claim 26, is characterized in that, described in provide the amino acid whose step of epi-position formative to comprise:

(a) select one group of conjugate, each conjugate comprises a base and tail base, and a basic matrix row, and wherein each stature base comprises amino acid;

(b) make them form non-covalent connection, wherein tail base hydrophobicity assemble and described conjugate is movably;

(c) detect between described non-covalent connection and target ligands and interact fully;

(d) the one group of conjugate repeating step (a) being optionally listed as with a basic matrix with modification is to (c); With