CN1897976A - Antagonists of the bradykinin b1 receptor - Google Patents

Abstract

The present invention relates to certain biologically active peptides and conjugated peptides which can be used as therapeutics or prophylactics against diseases or conditions linked to B1 as the causative agent. In a preferred embodiment of the invention biologically active PEG-conjugated peptides are provided. In one aspect of the present invention, pharmacologically active PEG-conjugated peptides of the present invention are useful to treat inflammation or pain.

Description

The bradykinin b 1 receptor antagonist

The application requires the U.S. Provisional Application No.60/513 of submission on October 22nd, 2003, the U.S. Provisional Application No.60/538 that on January 24th, 913 and 2004 submitted to, and 929 priority, these two applications are included this paper in as a reference.

Background of invention

At any given day, only just more than 200 ten thousand people are arranged because of chronic pain disability (T.M.Jessell in the U.S.; D.D.Kelly, pain and analgesia (Pain and Analgesia), PRINCIPLES OFNEURAL SCIENCE, the 3rd edition (T.M.Jessell compiles (1991) for E.R.Kandel, J.H.Schwartz).Unfortunately, at present for treatment of pain just part effectively, and a lot of treatment also have change lifestyles, weak and/or dangerous side effect.For example, nonsteroidal anti-inflammatory drug (" NSAIDs ") only has medium effect as aspirin, ibuprofen and indometacin to inflammatory pain, but they are toxic to kidney, and high dose is easy to cause gastrointestinal irritation, ulcer, hemorrhage, cardiovascular risk to increase and confusion of consciousness.Patient with the opioid treatment usually confusion of consciousness and constipation can occur, and secular opioid treatment is relevant with toleration and dependency.Local anesthetic such as lignocaine and mixelitine cause the forfeiture of normal threshold of feeling in inhibition of pain.In addition, when general was used, local anesthetic was relevant with the cardiovascular ill effect.Therefore, current aspect the treatment of chronic pain, still some need not be met.

Pain is a kind of consciousness, and it is based on the signal that receives from environment, and by nervous system transmission and explanation (summary is referring to Millan, M.J., the inducing of pain: scan .Prog Neurobiol 57:1-164 (1999)).Destructive stimulus as heat with contact the sensory receptors that makes the skin special secondary schoolization and transmit a signal to central nervous system (" CNS ").This process is called nociception, and the peripheral sensory neuron that mediates this process is a nociceptor.According to the intensity of the signal that transmits from nociceptor and CNS extraction and the processing to this signal, the people will or can not experience the pain of destructive stimulus.When a people's the pain sensation conformed to the intensity that stimulates rightly, pain played its original defencive function.Yet some tissue injurys cause a kind of phenomenon that is called hyperalgesia or short nociception (pronociception), wherein, because people's pain threshold has been lowered, so harmless relatively stimulation is perceived as violent pain.Inflammation and nerve injury all can cause hyperalgesia.Therefore, suffer from as sunburn, osteoarthritis, colitis, carditis; dermatitis, myositis, neuritis; inflammatory bowel, the people of collagen vascular disease (collagen vascular diseases) inflammatory condition such as (comprising rheumatic arthritis and lupus), its pain usually can strengthen.Similarly, the nerve injury that causes of wound, operation, amputation, abscess, causalgia, collagen vascular disease, demyelination, trigeminal neuralgia, cancer, chronic alcoholism, apoplexy, thalamic pain syndrome, diabetes, herpes infection, acquired immune deficiency syndrome (AIDS) (" AIDS "), toxin and chemotherapy all can cause pain excessive.

Because the mechanism to nociceptor conduction external signal under normal and hyperalgesia condition has had better understanding, can the related process of targeting hyperalgesia, come the reduction of inhibition of pain threshold value, thus the degree of minimizing pain.

Kallidin I (BK) and related peptides are the physiological action of kallidins (Lys-BK) (seeing Table 3) mediation kassinin kinin to cardiovascular and kidney system.Yet these bioactive peptide of BK and kallidins are degraded rapidly by the peptidase in blood plasma and other biological fluid, and are discharged from various kinds of cell, thereby it is reported that the half-life of BK in blood plasma is about 17 seconds (1).In health, BK and kallidins are by the rapid metabolism of carboxypeptidase N, and this enzyme is removed the arginine of carboxyl terminal, produce to remove the BK of arginineization (des-Arg) or go the blood vessel of arginineization to unfold element.Removing arginineization-kallidins is one of main kassinin kinin that is present in philtrum, the pathophysiology of kassinin kinin (pathphysiological) effect in its Mediated Human.Remove arginine BK or remove arginineization-kallidins except being the strong proinflammatory peptide, (summary is seen Regoli and Barabe for also known their induction of vascular diastoles, vascular permeability and bronchoconstriction, the pharmacology of Kallidin I and relevant kassinin kinin, pathology Reviews, 32 (1): l-46 (1980)).In addition, as if removing arginine BK and removing arginineization-kallidins is the mediation person who is even more important for inflammation and inflammatory pain, and participates in keeping of these processes.Also have a lot of evidences to show, in the disease such as septic shock, arthritis, angina pectoris and migraine that with pain are marked feature, go the generation of arginineization-kallidins excessive.

The membrane receptor of mediation kassinin kinin multiple-effect effect is two different types, is called B1 and B2.(Menke is etc., the expressivity clone .J.Biol.Chem.269:21583-21586 (1994) of people b1 bradykinin receptor to comprise philtrum clone and this receptor of two types that checked order from a lot of species; Hess etc., the clone of people's Kallidin I (BK-2) receptor and pathology are identified .Biochem.Biophys.Res.Commun.184,260-268 (1992)).They are typical g protein coupled receptors, and 7 membrane spaning domains of inferring are arranged.In various tissues, the BK receptor is coupled to each known second message,second messenger.The B2 receptor is the topmost form of bradykinin receptor seemingly, and it is higher to the affinity of BK.Basically, all normal physiological reaction and a lot of Pathophysiology logicality reactions to Kallidin I all are that B2 is receptor-mediated.

On the other hand, the B1 receptor is to going arginine BK with respect to the affinity to BK higher (seeing Table 3), and goes arginine BK not have activity for the B2 receptor.In addition, the B1 receptor is not expressed in the great majority tissue usually.Their expression is hurt or tissue injury and induced (Marceau, F. is etc., kassinin kinin B1 receptor: summary .Immunpharmacology, 30:1-26 (1995)) in the chronic inflammatory disease of some type or general wound.And, in rabbit, rat and pig, giving after lipopolysaccharide (LPS) or the inflammatory cytokine, the receptor-mediated reaction of B1 is raised (Marceau etc., (1998)) from zero level.

The pain inducing properties of kassinin kinin and the inducibility of B1 receptor are expressed and are made the B1 receptor become exploitation to make us interested target spot when directly specific effect is in wounded tissue and to the antiinflammatory of the effect minimum of normal structure, anti-nociceptive pain, antihyperalgesia and analgesic.Though identified the peptide antagonists of multiple targeting B1 receptor, but because they are organized degraded and the effectively kidney scavenging action very fast with the serum peptidase, these antagonisies are subjected to the restriction of its relatively poor effective half-life in the development aspect the therapeutic analgesic.Recently, proved that in vitro stability test having the substituent peptide analogues of alpha-non-natural amino acid has resistance to peptidase (summary is seen Regoli etc., bradykinin receptor and their antagonist .European Journalof Pharmacology, 348:1-10 (1998); Stewart, J.M., etc., brad ykinin antagonists: existing progress and vision of the future .Immunopharmacology, 43:155-161 (1999); And Stewart, J.M., etc., metabolic resistance brad ykinin antagonists: development and application .Biol.Chem., 382:37-41 (2001)).

Extensively approval, protein is the method for a kind of body-internal-circulation half-life of significant prolongation therapeutic protein with poly-(ethylene glycol) covalent coupling (PEG).PEGization mainly is to realize its effect (Zalipsky by postponing kidney scavenging action (because peg moiety has increased proteinic hydraulic radius greatly), S., Deng, utilize functionalized poly (ethylene glycol) modified polypeptide, poly-(ethylene glycol) chemical property: biotechnology and biological medicine are used, J.M.Harris compiles. (1992), Plenum Press:New York.p.347-370.).PEGization comprises the immunogenicity that increases its dissolubility, anti-Proteolytic enzyme degraded and reduce therapeutical peptide to proteinic other benefit.The advantage of PEGization comprises PEG-ADA Adenosine deaminase (Adagen from several PEGization albumen ^TM/ Enzon Corp.), PEG-L-asparaginase (Oncaspar ^TM/ Enzon Corp.), PEG-Interferon Alpha-2b (PEG-Intron ^TM/ Schering/Enzon), PEG-Intederon Alpha-2a (PEGASYS ^TM/ Roche) and PEG-G-CSF (Neulasta ^TM/ commercialization Amgen) and the medicine of other a lot of clinical experimental stages confirm.On the other hand, the PEGization of little therapeutic peptide has special difficult point, also is not widely used.One of obstacle of maximum is must guarantee to have kept biologic activity in the final conjugate in the peptide PEGization.Because therapeutic peptide usually only comprises active required minmal sequence, so they are often very little, so do not tolerate replacement comparatively speaking.With respect to these peptides itself, peg moiety is disproportionately big, therefore might spatially disturb peptide more: the specificity of receptors bind interacts, and this interaction is active required.So the ability that peptide tolerance PEGization also still maintains with enough specific activities of medicine is very unpredictable; must determine (Morpurogo through checking; Deng, with the α and the ε amino group of PEG selectivity alkanisation and a kind of somatostatin analogue of acidylate: particular chemical is used to optimize biological conjugate (Tailored Chemistry for Optimized Bioconjugates) .Bioconjugate Chem.13:1238-1243 (2002)).

Obviously, need new, safety and effectively treat the method for inflammation and pain.It is favourable that a kind of like this B1 specific peptide antagonist is provided, by cycle period (postponing to remove), dissolubility, the stability that increases B1 specific peptide antagonist, and/or reduce the immunogenicity of this molecule, obtain in therapeutic process, can in health, have better toleration.The circulating half-life increase will make administration frequency reduce, and administration frequency reduces and will make doctor and patient all convenient, be particularly useful for the patient of automedication.Other benefit that administration frequency reduces comprises that the medicine that gives the patient reduces and compliance increases.

Summary of the invention

Therefore, an object of the present invention is to provide the novel of B1 receptor in conjunction with preparation, compare with known B1 antagonist, these preparations have clear and definite good pharmacokinetic properties in vivo, and the abundant activity of antagonism B1 receptor still, thereby can be used for treatment or prevent following disease: the disease of inflammation, pain and other B1 mediation includes but not limited to asthma and allergic rhinitis.New type of peptides antagonist form that these medicines provided by the invention are B1 receptors and link coupled peptide antagonists form.In one embodiment, novel B 1 receptor peptide antagonists of the present invention comprises the aminoacid sequence shown in SEQ ID NO:15-54 arbitrary.

According to certain embodiments of the present invention, L-aminoacid or amino acid whose one or more (the preferred 1-9 kind) amino acid residues of stereomeric D-that are independently selected from 20 kinds of genetic codings are coupled to the one or both ends of the peptide sequence shown in the SEQ ID NO:15-54.

In another embodiment, the present invention also provides link coupled peptide, compare with known B1 peptide antagonists, these link coupled peptides have clear and definite good pharmacokinetic properties in vivo, thereby and still fully in conjunction with also antagonism B1 receptor active can application in treatment.

One aspect of the present invention comprises the coupling peptide of formula (I):

F-[(X ¹)-(Y ¹) _n] I

Wherein:

F is covalently bonded in X ¹Or Y ¹Carrier (preferred F be the peg moiety or derivatives thereof);

X ¹And Y ¹Independent respectively separately is formula-L ¹-P ¹With-L ²-P ²Peptide;

L ¹And L ²Independent separately is joint;

N is 0-3; With

P ¹And P ²Independent separately is the peptide antagonists of bradykinin b 1 receptor.Preferred P ¹And P ²Comprise aminoacid sequence and the derivant thereof shown in SEQ ID NO:5-26,43-60 arbitrary.

Another aspect of the present invention provides a kind of pharmaceutical composition, and said composition comprises the excipient carrier material, is dispersed with coupling peptide of the present invention in described excipient carrier material.

Another aspect of the present invention provides a kind of curative Therapeutic Method, and this method comprises the compositions that comprises excipient and at least a peptide of the present invention and/or coupling peptide that the mammal of these needs pharmacy effective dose is arranged.

Peptide of the present invention and/or coupling peptide have treatment owing to the treatment of diseases that the B1 activation mediates is worth, the described disease that mediates owing to the B1 tongueization includes but not limited to, chronic pain and inflammation, septic shock, arthritis, osteoarthritis, osteoarthritis, angina pectoris, asthma, allergic rhinitis and the migraine of inflammatory and nerve origin.

Can this patient such as people (or other mammal) of needing be arranged with peptide of the present invention and/or coupling peptide and suitable pharmaceutical carriers material fit and with effective dose, thereby with peptide of the present invention and/or being used for the treatment of property of coupling peptide or preventative purpose.

Can carry out conservative by aminoacid sequence and modify, obtain other useful peptide and/or coupling peptide peptide as herein described and/or carrier-coupling peptide.The peptide that the conservative modification is produced and/or function, physics and the chemical characteristic of coupling peptide and adorned peptide and/or coupling peptide are similar.These conservative modification of peptide described herein and/or coupling peptide also are thought of as one embodiment of the present invention.

The present invention relates to a kind of method for preparing coupling peptide described herein on the other hand, and the method comprising the steps of:

To have the chemical compound and carrier (F) reaction of following structure, generation has formula F-[(X ¹)], F-[(X ¹)]-F, F-[(X ¹)-(Y ¹) _N], F-(X ¹)-(Y ¹) _NOr F-(X ¹)-(Y ¹) _NThe coupling peptide of-F:

(X ¹)-(Y ¹) _N

Wherein,

X ¹And Y ¹Independent respectively separately is formula-L ¹-P ¹With-L ²-P ²Peptide;

L ¹And L ²Independent separately is joint;

N is 0-3; With

P ¹And P ²Independent separately is the peptide antagonists of bradykinin b 1 receptor.

Preferred F is the peg moiety or derivatives thereof.More preferably, P ¹And P ²Independent of separately comprising the peptide antagonists of the bradykinin b 1 receptor of peptide sequence shown at least one SEQ ID NO:5-60.More preferably, X ¹It is the peptide shown in the SEQ IDNOS:27-41.After considering following detailed description of the present invention, other aspects and advantages of the present invention will be very obvious.

Detailed Description Of The Invention

The present invention is based on an astonishing discovery, promptly, can will it is generally acknowledged that a class peptide that does not tolerate very much replacement carries out aminoacid replacement and/or N-terminal is coupled to various carriers at N-terminal, with peptide and/or the peptide conjugate that provides usefulness, these peptides and/or peptide conjugate are compared with known similar peptide, its effectiveness is very lasting, thereby can be used for treating inflammation and pain.Therefore peptide of the present invention and/or peptide conjugate provide huge treatment benefit with respect to known B1 peptide antagonists.More particularly, the present inventor finds, by replacing the N terminal amino acid of known B1 peptide antagonists, and/or determine that with having size and peptidyl or the non-peptidyl joint formed are coupled to carrier (such as, but be not limited to Polyethylene Glycol (PEG) molecule) with these peptide antagonists, some previously described shortcomings during the therapeutic that can overcome known B1 peptide antagonists is used are farthest kept the activity of antagonist and specificity in effective half-life in prolonging the antagonist body.(perhaps) in addition, compare with the link coupled peptide conjugate of less PEG polymer although find, though reduce a little with big link coupled its external activity of B1 peptide antagonists of PEG polymer, the circulating half-life that this big PEG-conjugate prolongs makes its time of staying in vivo and renders a service greatly and increases.In addition, the inventor finds that also the PEG bulk of molecule that is connected in B1 receptor peptide antagonists is a key factor of optimizing effective half-life in inherent activity of antagonist and the body thereof.For example, in the dependent body inner model of pain, prove a kind of acetylizad B1 peptide antagonists can reach 4 hours behind multiple dosing effectiveness.It is shocking after single agent fast injection, to have up to 2 days respectively and at least 4 days effectiveness with 5kD and the link coupled identical peptide of 20kD PEG molecule in mode described herein.

Before describing peptide of the present invention and carrier or the link coupled bradykinin b 1 receptor peptide antagonists of PEG, its preparation and using method, should understand, the method of peptide involved in the present invention and/or link coupled peptide antagonists the invention is not restricted to described concrete peptide and/or link coupled peptide antagonists, because can have a little variation.Should be understood that term used herein just for the purpose of describing specific implementations, rather than restrictive, because scope of the present invention only is limited to the appended claims.

Consideration includes but not limited to purpose described herein and mode and the link coupled bradykinin b 1 receptor binding peptide of carrier, novel B 1 binding peptide antagonist as herein described and B1 binding peptide antagonist known in the art, include but not limited to, an arbitrary piece of writing disclosed any peptide: Regoli etc. of following publication (all including this paper in as a reference in full), bradykinin receptor and their antagonist .Eur.J.of Pharma., 348:1-10 (1998); Neugebauer, W., etc., the B1 kinin receptor antagonist .Can.J.Physiol.Pharmacol. with multienzyme resistance, 80:287-292 (2002); Stewart, J.M., etc., brad ykinin antagonists: existing progress and vision of the future .Immunopharmacology, 43:155-161 (1999); Stewart, J.M., etc., metabolic resistance brad ykinin antagonists: development and application .Biol.Chem., 382:37-41 (2001); PCT publication WO98/07746; And US Patent No: 4,693,993,4,801,613,4,923,963,5,648,336,5,834,431,5,849,863,5,935,932,5,648,333,5,385,889,5,444,048 and 5,541,286.

Unless qualification is arranged under the particular case in addition, otherwise following qualification of the employed term of whole description.

In three kinds of modes the natural amino acid residue is described: the trigram of aminoacid full name, standard or the single-letter of standard (as shown in the table).

A＝Ala G＝Gly M＝Met S＝Ser

C＝Cys H＝His N＝Asn T＝Thr

D＝Asp I＝Ile P＝Pro V＝Val

E＝Glu K＝Lys Q＝Gln W＝Trp

F＝Phe L＝Leu R＝Arg Y＝Tyr

Unless expressly stated otherwise,, otherwise natural herein and alpha-non-natural amino acid comprises amino acid whose D type-and L type-isomer.The abbreviation of alpha-non-natural amino acid used herein and U.S. Patent No. 5,834,431, PCT publication WO 98/07746 and Neugebauer wait in (2002) equally, and these documents are all included this paper in as a reference in full.In addition, abbreviation " Dab " and " D-Dab " refer to respectively the L type of alpha-non-natural amino acid D-2-aminobutyric acid-with D type-isomer.Abbreviation " 3 ' PaI " and " D-3 ' PaI " refer to the L-and the isomer of D-type of alpha-non-natural amino acid 3 '-pyridine radicals alanine respectively.Abbreviation " Igl " comprises " Igla " and " Iglb " (being respectively α-(1-indanyl) glycine and α-(2-indanyl) glycine).Similarly, " DIgl " comprises " D-Igla " and " D-Iglb " (being respectively the D type-isomer of α-(1-indanyl) glycine and α-(2-indanyl) glycine).Preferably, when using in this article, Igl is Iglb, and D-Igl is D-Iglb.

" carrier-link coupled peptide " or " link coupled peptide " refer to the chemical compound that has biologic activity and therapeutic effect is provided after giving mammal.These two parts comprise (1) at least a B1 peptide antagonists and (2) at least a following limit and the residue of described peptide itself or with peptidyl or the covalently bound carrier of non-peptidyl joint (including but not limited to the aromatic radical joint) (the residue covalent bond of these joints and described peptide).

" the link coupled peptide of PEG " or " peptide of PEGyization " refer to a kind of two-part chemical compound, and it has biologic activity and therapeutic effect is provided after giving mammal.Described two parts comprise (1) at least a B1 peptide antagonists and (2) at least a and described peptide itself residue or with peptidyl or the covalently bound polyalkylene glycol moiety of non-peptidyl joint (including but not limited to the aromatic radical joint) (the residue covalent bond of these joints and described peptide).

" Polyethylene Glycol " or " PEG " refers to ployalkylene glycol chemical compound or derivatives thereof, it has or does not have bonded material, do not derive or derive with combination or activatory part (for example, mercaptan, triflate (triflate), trifluoro esilate (tresylate), aziridine, oxirane, adjacent pyridyl disulfide, vinyl sulfone(Remzaol, iodoacetamide or maleimide amine moiety).

PEG is the water-soluble polymer of knowing, and can buy or with method well known in the art (Sandler and Karo, poly-mer Synthesis by commercial sources, Academic Press, New York, Vol.3,138-161 page or leaf) ring-opening polymerisation by ethylene glycol prepares.Among the application, its broad sense got in term " PEG ", comprises any list-or the peg molecule of multifunctional form, no matter its size or have or not at the PEG end is modified, PEG can represent by following formula:

X-O(CH ₂CH ₂O) _n-1CH ₂CH ₂OH， II

。

Wherein n is 20-2300, and X is H or end modified, as C _1-4Alkyl.

Preferably, PEG one end used among the present invention is hydroxyl or methoxyl group, and promptly X is H or CH ₃(" methoxyl group PEG ").Note, work as X=CH ₃The time, the other end of PEG (is OH suc as formula end shown in the II) is covalently bound by ether-oxygen bond and activated partial.When X=H,, the two ends of PEG produce linear Bifunctionalized PEG thereby all being connected in activated partial by ehter bond.When being used for chemical constitution, term " PEG " comprises following formula II (the not hydrogen of signify hydroxy), and oxygen wherein can react with the free carbon atom of joint to form ehter bond.More particularly, for PEG is connected with peptide, PEG must be " activation " form.Activatory PEG can represent by formula III:

(PEG)-(A) III

Wherein, the covalently bound formation ehter bond of carbon atom of PEG (definition as mentioned above) and activated partial (A), and (A) contain can with amino, imino group or mercapto groups reactive activity group on the peptide ammino acid residue or blank area reactive activity group that can be covalently bound with this peptide.

The method for preparing activatory PEG is well known in the art, for example, sees U.S. Patent No. 5,643,575,5,919,455,5,932,462 and PCT publication WO 95/06058 (all in full include this paper in as a reference).Can prepare suitable activatory PEG with a lot of conventional reactions.For example, can be according to Buckmann and Merr, Makromol.Chem., the method of 182:1379-1384 (1981), with PEG-monomethyl ether (can buy from Union Carbide) and N, N '-dicyclohexyl carbodiimide (DCC) and N-hydroxy-succinamide (NHS) reaction prepare the N-hydroxy-succinamide ester (M-NHS-PEG) of PEG.Other activatory PEG as PEG-aldehyde, can obtain from commercial channels, for example, Nektar Therapeutics (Huntsville, A1) or Enzon, Inc. (Piscataway, N.J.).The example that is used for the preferred activated PEG of the object of the invention is PEG-propionic aldehyde and PEG-butyraldehyde, can (Huntsville A1) buys from Nektar Therapeutics.The structural formula of PEG-propionic aldehyde is PEG-CH ₂CH ₂CHO is described in U.S. Patent No. 5,252,714, and document integral body is included this paper in as a reference.In addition, Bifunctionalized PEG aldehyde can be used for preparing the dimer conjugate.

The reactive PEG of other preferred amine comprise methoxyl group-PEG succinyl phosphorons amino propyl acid salt (ester) (mPEG-SPA) and methoxyl group-PEG butanimide butyrate (ester) (mPEG-SBA), mPEG-benzotriazole carbonate (ester) or mPEG-p-nitrobenzophenone carbonate (ester), they can be available from Nektar Therapeutics (Huntsville, A1), Enzon, Inc. (Piscataway, N.J.) or NOF Corporation (Tokyo, Japan), there is various molecular weight to select.Other preferred activated PEG partly comprises thiol-reactive functional group, includes but not limited to, PEG vinyl sulfone(Remzaol, its structural formula are PEG-CH ₂CH ₂SO ₂-CH=CH ₂, mPEG-iodoacetate and mPEG-thioester as follows:

The MPEG-thioesters

The MPEG-iodoacetamide

Other the preferred activated PEG that is used to produce PEG coupling peptide of the present invention is the PEG-maleimide.For preparation PEG coupling peptide of the present invention, maleimide mono methoxy PEG is particularly useful.

The preferred activated PEG that is used to produce PEG coupling peptide of the present invention is the multivalence PEG that activation residue more than is arranged.Preferably the multivalence peg moiety includes but not limited to as follows these:

According to actual needs, can adopt the PEG of any molecular weight, for example, about 1,000 dalton (Da)-100,000Da (n is 20-2300).Number of repeating units n approaches the molecular weight represented with dalton among the PEG.The molecular weight that PEG on the preferred activation joint combines is suitable for pharmaceutical applications.Therefore, the combination molecule amount of PEG molecule should not surpass 100,000Da.

Preferably, the combination molecule amount or the total molecular weight that are used for the PEG of PEG coupling peptide of the present invention are about 3,000Da-60, and 000Da (total n is 70-1,400) is more preferably 8,800Da-36,000Da (total n is about 200-820).Most preferred PEG combination molecule amount is about 20,000Da-24,000Da (total ni is about 450-540).

Other ployalkylene glycol chemical compound as polypropylene glycol, can be used for the present invention.Other suitable ployalkylene glycol chemical compound includes but not limited to, the electrically charged or neutral polymer of following type: glucosan, poly-acetylneuraminic acid ammonia or other polymer based on carbohydrate, amino acid polymer, and biotin derivative.

Term " comprises " and refers to that peptide or coupling peptide can comprise other molecular moiety at the N-of given sequence or the one or both ends of C-end, include but not limited to aminoacid.Certainly, these other molecular moieties should significantly not disturb the activity of peptide or coupling peptide.

Term used herein " native peptides " refers to not link coupled B1 peptide antagonists described herein or known in the art.

Term " is derived ", " derivant " or " or deutero-" comprises the peptide or the coupling peptide of process and generation respectively, and wherein, (1) peptide or coupling peptide have annulus, for example, and crosslinked in the coupling peptide between the cysteine residues; (2) peptide or coupling peptide are crosslinked or have crosslink sites, and for example, thereby peptide or coupling peptide have cysteine residues to form crosslinked dimer in cultivation or in the body; (3) have-NH ₂N-terminal quilt-the NRR of the coupling peptide of end group ¹, NRC (O) R ¹,-NRC (O) OR ¹,-NRS (O) ₂R ¹,-NHC (O) NHR, butanimide group, replacement or unsubstituted benzyloxycarbonyl group-NH-replace, wherein R and R ¹And cyclic substituents below this paper definition; (5) C-terminal quilt-C (O) R ²Or-NR ³R ⁴Replace, wherein R ², R ³And R ⁴Such as below this paper definition; (6) link coupled peptide, wherein, independent amino acid moiety is with modifying with the material of selected side chain or terminal residue reaction.Further describe derivant below this paper.

Term " B1 " refers to bradykinin b 1 receptor (seeing Judith M Hall, BK receptor summary .Pharmac.Ther.56:131-190 (1992)).Except specify in addition, B1 or bradykinin b 1 receptor refer to people's bradykinin b 1 receptor (hB1).Preferably, hB1 is a wild-type receptor.Preferred, hB1 is the bradykinin receptor described in the GenBank accession number AJ238044.

Term used herein " peptide " refers to that molecular weight is a 4-40 amino acid whose molecule, preferred 10-20 amino acid whose molecule, most preferably 15-18 amino acid whose molecule.Term used herein " dipeptides " refers to have two amino acid whose molecules.Term " tripeptides " refers to have three amino acid whose molecules.

The structural analysis of protein-protein interaction also can be used for illustrating simulation large protein aglucon in conjunction with active peptide.In this analysis, crystal structure can illustrate the character and the relative orientation of the Key residues of albumen aglucon, thus peptide like the design class in view of the above.See, for example, Takasaki etc., Nature Biotech., the 15th volume, 1266-1270 page or leaf (1997).Also available these analytical methods are studied the interaction of receptor protein and peptide of the present invention, carrier-coupling peptide or PEG coupling peptide, thereby can further modify described peptide and peptide conjugate in view of the above, increase its binding affinity.

Term used herein " effective dose " and " treatment effective dose " are when being used to describe the link coupled B1 peptide antagonists of peptide, carrier-link coupled, peptide or PEG, refer to be enough to produce the amount or the dosage of required effect (that is, treating) with peptide of the present invention, carrier-link coupled peptide or the link coupled B1 peptide antagonists of PEG.In the present invention, required result for example is alleviating of required inflammation and/or pain, or can be observed the reduction of one or more level of biological activity of B1.More particularly, the treatment effective dose is meant that interior peptide of a period of time and/or coupling peptide are enough to suppress and disease described herein such as inflammation or the relevant amount of the pathological process of definition clinically of pain in the patient who carries out interior therapeutic with this medicine.This effective dose can change according to selected concrete peptide and/or coupling B1 peptide antagonists, also can change according to the several factors relevant with treatment target, situation and disease seriousness.For example, if described peptide and/or link coupled B1 peptide antagonists vivo medicine-feeding, then patient's age, body weight and health status and the dose-effect curve that obtains from the pre-clinical trial of animal and toxicity data are all at the row of consideration.If described medicine, then also will design a lot of pre-clinical in vitro studies in external and cells contacting and estimate some parameters, as absorption, half-life, dosage, toxicity etc.A kind of given effective amount of drug or treatment effective dose fix within the limit of power of persons skilled in the art really.

Term " pharmacological activity " refers to that described material is confirmed as having the activity of the drug parameters of influence or morbid state (for example, pain).In the present invention, this term is often referred to disease or unusual medical condition or the disease that B1 is inductive or B1 mediates, more particularly, and the antagonism of dactylitis disease or pain.

Term " antagonist ", " inhibitor " and " anti-gaonist " are (for example, see, Rianne A-F.de Ligt, Deng, British Journal of Pharmacology 2000,130,131) refer to prevent, hinder, reduce, reduce or disturb in some way the molecule of the biologic activity of interested associated protein.The present invention's preferred " antagonist " or " inhibitor " be in the active in vitro tests of B1 in conjunction with and suppress B1, IC ₅₀Be 500nM or molecule still less.The present invention's preferred " antagonist " or " inhibitor " be in the active in vitro tests of B1 in conjunction with and suppress B1, IC ₅₀Be 100nM or molecule still less.The present invention's most preferred " antagonist " or " inhibitor " be in the active in vitro tests of B1 in conjunction with and suppress B1, IC ₅₀For 50nM or still less and prevention, alleviation or eliminate pain (detecting) and/or in the animal body inner model of edema, inflammation or pain, suppress the biochemical molecule of attacking as at least a generally accepted animal pain body inner model.

In addition, the physiologically acceptable salt of peptide of the present invention or coupling peptide also contained in this paper.Phrase used herein " physiologically acceptable salt " and " the last acceptable salt of pharmacology " are interchangeable, comprise that known or later discovery is any salt of pharmaceutically acceptable (can be used for the treatment of Homoiotherm).Some object lessons comprise: acetate, trifluoroacetate, halogen acid salt example hydrochloric acid salt and hydrobromate, sulfate, citrate, tartrate, glycollate, oxalates, mineral acid and organic acid salt, these acid include but not limited to, hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methanesulfonic acid, ethyl sulfonic acid, malic acid, acetic acid, oxalic acid, tartaric acid, citric acid, lactic acid, fumaric acid, succinic acid, maleic acid, salicylic acid, benzoic acid, phenylacetic acid, mandelic acid etc.When The compounds of this invention comprises acidic functionality such as carboxyl, to being that persons skilled in the art are known, comprise alkali metal, alkaline-earth metal, ammonium, quaternary ammonium cation etc. for the suitable pharmaceutically acceptable cation of carboxyl.Other example of " the last acceptable salt of pharmacology " as follows and Berge etc., J.Pharm.Sci.66:1 (1977).

" blocking group " refers generally to well known in the artly be used to prevent that selecteed reactive group such as carboxyl, amino, hydroxyl, sulfydryl etc. from carrying out the group of unnecessary reaction (as nucleophilic, electrophilic, oxidation, reduction reaction etc.).Preferred blocking group is a suitable blocking group as herein described.The example of amido protecting group includes but not limited to, the pi-allyl of the cyclisation alkenyl alkyl of the aralkyl of aralkyl, replacement, cyclisation alkenyl alkyl and replacement, pi-allyl, replacement, acyl group, alkoxy carbonyl, aromatic alkoxy carbonyl, silicyl etc.The example of aralkyl includes but not limited to, benzyl, neighbour-methyl-benzyl, trityl and benzhydryl, and they can be by optional replacements such as halogen, alkyl, alkoxyl, hydroxyl, nitro, amide groups, acyl groups, and salt, as  salt, ammonium salt.The example of aryl comprises phenyl, naphthyl, indanyl, anthryl, 9-(9-phenyl fluorenyl), phenanthryl, durene base etc.The cyclisation alkylidene alkyl (cycloalkylenylalkyl) of cyclisation alkenyl alkyl or replacement preferably has the 6-10 carbon atom, includes but not limited to cyclohexenyl group methyl etc.Suitable acyl group, alkoxy carbonyl and aromatic alkoxy carbonyl comprise benzoyl, bytyry, acetyl group, trifluoroacetyl group, tribromo-acetyl base, phthalyl of benzyloxycarbonyl group, tertbutyloxycarbonyl, isobutyl boc, benzoyl, replacement etc.The mixture of available blocking group is protected same amino, and for example primary amine group can be protected with aralkyl and aromatic alkoxy carbonyl.The nitrogen that the amido protecting group also can be connected with them forms heterocycle; for example; 1, two (methylene) benzene of 2-, phthalimide-based, succinimido, succinimide base etc., these heterocyclic groups also can further comprise aryl and the cycloalkyl ring that adjoins.In addition, these heterocycles can be single, double or trisubstituted, for example the nitro phthalimide-based.Also can protect amino in case unnecessary reaction takes place, as oxidation reaction by for example forming addition salts with hydrochloric acid, toluenesulfonic acid, trifluoroacetic acid etc.A lot of amido protecting groups also are suitable for protecting carboxyl, hydroxyl or sulfydryl.For example, aralkyl.Alkyl, for example the tert-butyl group also is the proper group of protection hydroxyl and sulfydryl.

Silicyl is by the optional silicon atom that replaces of one or more alkyl, aryl and aralkyl.Suitable silicyl blocking group includes but not limited to trimethyl silyl, triethylsilyl, triisopropyl silicyl, t-butyldimethylsilyl, 3,5-dimethylphenyl silicyl, 1; two (dimetylsilyl) benzene, 1 of 2-, two (dimetylsilyl) ethane of 2-and diphenyl methyl silicyl.That the silylanizing of amino group provides is single-or the bis-silyl amino group.The silylanizing of (aS)-2-Amino-5-chloro-a-(cyclopropylethynyl)-a-(trifluoromethyl)benzenemethanol. can produce N, N, O-three-silicyl derivant.Silyl functional group is removed and can be passed through at an easy rate from silyl ether functional group, and for example, (in situ) finishes with metal hydroxides or ammonium fluoride agent treated on the spot in an independent reactions steps or when reacting with alcohol.Suitable sillylation reagent is the combination product of trimethylsilyl chloride, the tert-butyl group-dimetylsilyl chlorine, diphenyl methyl silyl chloride or they and imidazoles or DMF.Is well known to those skilled in the art with the silylated method of amine with the method for removing the silicyl blocking group.The method for preparing these amine derivatives from corresponding aminoacid, amino acid amide or amino-acid ester is that organic chemistry filed comprises that the technical staff of aminoacid/amino-acid ester or amino alcohol chemical field knows.

Under the situation that does not influence the molecule other parts, blocking group is removed.These methods are well known in the art, comprise acid hydrolysis, hydrogenolysis etc.Preferable methods comprises, suitable solvent system as alcohol, acetic acid etc. or its mixture in, blocking group such as benzyloxycarbonyl group group are removed by hydrogenolysis with the palladium on the carbon.Can be at suitable solvent system, as in two  alkane or the METHYLENE CHLORIDE with removing the tert-butyl group-carbonyl-protection group in inorganic or organic acid such as HCl or the trifluoroacetic acid.The amide that can easily neutralize and produce obtains free amine.Can under hydrolysis well known to those skilled in the art and hydrogenolysis condition, remove carboxy protective group, as methyl, ethyl, benzyl, the tert-butyl group, 4-methoxybenzene ylmethyl etc.

It should be noted that chemical combination of the present invention can comprise the group that may exist with tautomeric form, the heteroaryl that replaces as ring-type or non-annularity amidino groups and guanidine radicals, hetero atom (Y '=O, S, NR) etc., in the following example shown in:

Though this paper only names, describes, demonstration and/or claimed a kind of form, this name, description, demonstration and/or claimed form itself have comprised all tautomeric forms.

The present invention also considers the prodrug of The compounds of this invention.Prodrug is activity or non-active compound, by body physiological effect such as hydrolysis, metabolism etc. it is carried out chemical modification, makes prodrug become chemical compound of the present invention after giving the patient.The fitness and the technology of preparation and use prodrug are well known to those skilled in the art.See Svensson and Tunek for the describe, in general terms of the prodrug that comprises ester, drug metabolism summary, 165 (1988) and Bundgaard, drug design, Elsevier (1985).The example of protected (masked) carboxylate anion comprises a lot of esters, as with alkyl (for example, methyl, ethyl), cycloalkyl (for example; cyclohexyl); the ester that aralkyl (for example, benzyl, to methoxy-benzyl) and alkyl-carbonyl oxoalkyl group (for example, valeryl oxo methyl) form.Amine is protected to be the methyl substituted derivant of aryl carbonyl oxo, and it is discharged free medicine and formaldehyde (Bundgaard J.Med.Chem.2503 (1989)) by the esterase cutting in vivo.Equally, contain acid NH group, for example medicines such as imidazoles, acid imide, indole are protected (Bundgaard, drug design, Elsevier (1985)) by N-acyl group oxo methyl.Hydroxyl is protected to be ester and ether.EP 039,051 (Sloan and Little, 4/11/81) discloses mannich base hydroxamic acid prodrug, and preparation and use.

The structure of coupling peptide

Summation.Carrier of the present invention-coupling peptide can be represented by the formula:

F-[(X ¹)-(Y ¹) _n] (IV)

Wherein:

X ¹And Y ¹Independent separately respectively is formula-L ¹-P ¹With-L ²-p ²Peptide;

F is covalent bond X ¹Or Y ¹Carrier;

L ¹And L ²Do not exist or independent separately for having the joint of 0-9 amino acid residue;

N is 0-3; With

P ¹And P ²If (existence) is independent separately to be the bradykinin b 1 receptor peptide antagonists.

The carrier of formula IV-its preferred implementation of coupling peptide is P ¹And P ²If (existence) is independent of separately having the bradykinin b 1 receptor peptide antagonists of arbitrary peptide sequence or derivatives thereof shown in the SEQ ID NO:5-60.

Other preferred implementation of carrier-coupling peptide comprises carrier-coupling peptide, wherein P of formula IV ¹And P ²If (existence) is defined as following formula:

NH ₂-a ⁰a ¹a ²a ³a ⁴a ⁵a ⁶a ⁷a ⁸a ⁹a ¹⁰a ¹¹a ¹²a ¹³a ¹⁴-COOH

Wherein:

a ⁰Be alkalescence or neutral aromatics, aliphatic, heterocycle or alicyclic aminoacid, the dipeptides that one or two residue that has basic side chain is arranged or tripeptides, or do not exist;

a ¹, a ², a ³And a ⁴Independent separately is alkalescence or neutral aromatics, aliphatic, heterocycle or alicyclic aminoacid;

a ⁶Be Ser;

a ⁵, a ⁷And a ⁸Independent separately is aromatics, aliphatic, heterocycle or alicyclic aminoacid, and condition is a ⁵, a ⁷And a ⁸At least one be selected from Chg, Cpg, Igla, Iglb, Niga and the Nigb of D-or L-configuration;

a ⁹, a ¹⁰, a ¹¹, a ¹², a ¹³And a ¹⁴Do not exist or independently separately be any natural amino acid.

Preferred, P ¹And P ²If (existence) is defined as following formula:

NH ₂-a ⁰a ¹a ²a ³a ⁴a ⁵a ⁶a ⁷a ⁸a ⁹a ¹⁰a ¹¹a ¹²a ¹³a ¹⁴-COOH

Wherein:

a ⁰Do not exist, or basic amino acid, the dipeptides that one or two is with the residue of basic side chain contained;

a ¹It is basic amino acid;

a ²Be Pro;

a ³Be Hyp;

a ⁴Be Gly;

a ⁵And a ⁸Be indanyl aminoacid;

a ⁶Be Ser;

a ⁷Be D-indanyl aminoacid;

a ⁸Be Cpg; With

a ⁹, a ¹⁰, a ¹¹, a ¹², a ¹³And a ¹⁴Do not exist or independently separately be any natural amino acid.

Preferred, P ¹And P ²If (existence) is defined as following formula:

NH ₂-a ⁰a ¹a ²a ³a ⁴a ⁵a ⁶a ⁷a ⁸a ⁹a ¹⁰a ¹¹a ¹²a ¹³a ¹⁴-COOH

Wherein:

a ⁰Do not exist, or basic amino acid, contain the dipeptides of one or two basic side chain;

a ¹It is basic amino acid;

a ²Be Pro;

a ³Be Hyp;

a ⁴Be Gly;

a ⁵Be Cpg;

a ⁶Be Ser;

a ⁷Be DTic;

a ⁸Be Cpg; With

a ⁹, a ¹⁰, a ¹¹, a ¹², a ¹³And a ¹⁴Do not exist or independently separately be any natural amino acid.

Preferred, carrier of the present invention-coupling peptide comprises carrier-coupling peptide of formula IV, wherein n=0 and X ¹Be to be selected from the peptide shown in the SEQ ID NO:27-41 and the peptide of derivant thereof.

The present invention also provides combination and the active PEG coupling of antagonism bradykinin b 1 receptor (B1) peptide, compares with not link coupled B1 peptide antagonists, and it has good pharmacokinetic properties clearly.PEG coupling peptide of the present invention can be represented with following formula V:

F-[(X ¹)-(Y ¹) _n] V

Wherein:

X ¹And Y ¹Independent separately respectively is formula-L ¹-P ¹With-L ²-P ²Peptide;

F is covalent bond X ¹Or Y ¹Peg moiety;

L ¹And L ²Do not exist or independent separately for having the joint of 0-9 amino acid residue;

N is 0-3; With

P ¹And P ²If (existence) is independent separately to be the bradykinin b 1 receptor peptide antagonists.

Its preferred implementation of PEG coupling peptide of formula V is P ¹And P ²If (existence) is independent of separately having the bradykinin b 1 receptor peptide antagonists of arbitrary peptide sequence shown in the SEQ ID NO:5-60 and its derivant.

Other preferred implementation of PEG coupling peptide comprises the PEG coupling peptide of formula V, wherein P ¹And P ²If (existence) is defined as following formula:

NH ₂-a ⁰a ¹a ²a ³a ⁴a ⁵a ⁶a ⁷a ⁸a ⁹a ¹⁰a ¹¹a ¹²a ¹³a ¹⁴-COOH

Wherein:

a ⁰Do not exist, or alkalescence or neutral aromatics, aliphatic, heterocycle or alicyclic aminoacid, alkaline dipeptides or tripeptides;

a ¹, a ², a ³And a ⁴Independent separately is alkalescence or neutral aromatics, aliphatic, heterocycle or alicyclic aminoacid;

a ⁶Be Ser;

a ⁵, a ⁷And a ⁸Be aromatics, aliphatic, heterocycle or alicyclic aminoacid, condition is a ⁵, a ⁷And a ⁸At least one be selected from Chg, Cpg, Igla, Iglb, Niga and the Nigb of D-or L-configuration; With

a ⁹, a ¹⁰, a ¹¹, a ¹², a ¹³And a ¹⁴Do not exist or independently separately be any natural amino acid.

Preferred, P ¹And P ²If (existence) is defined as following formula:

NH ₂-a ⁰a ¹a ²a ³a ⁴a ⁵a ⁶a ⁷a ⁸a ⁹a ¹⁰a ¹¹a ¹²a ¹³a ¹⁴-COOH

Wherein:

a ⁰Do not exist, or basic amino acid, alkaline dipeptides;

a ¹It is basic amino acid;

a ²Be Pro;

a ³Be Hyp;

a ⁴Be Gly;

a ⁵And a ⁸Be indanyl aminoacid;

a ⁶Be Ser;

a ⁷Be D-indanyl aminoacid;

a ⁸Be Cpg; With

a ⁹, a ¹⁰, a ¹¹, a ¹², a ¹³And a ¹⁴Do not exist or independently separately be any natural amino acid.

Preferred, P ¹And P ²If (existence) is defined as following formula:

NH ₂-a ⁰a ¹a ²a ³a ⁴a ⁵a ⁶a ⁷a ⁸a ⁹a ¹⁰a ¹¹a ¹²a ¹³a ¹⁴-COOH

Wherein:

a ⁰Do not exist, or basic amino acid, alkaline dipeptides;

a ¹It is basic amino acid;

a ²Be Pro;

a ³Be Hyp;

a ⁴Be Gly;

a ⁵Be Cpg;

a ⁶Be Ser;

a ⁷Be DTic;

a ⁸Be Cpg; With

a ⁹, a ¹⁰, a ¹¹, a ¹², a ¹³And a ¹⁴Do not exist or independently separately be any natural amino acid.

Preferred, PEG coupling peptide of the present invention comprises the PEG coupling peptide of formula V, wherein n=0 and X ¹It is the peptide that contains aminoacid shown in the SEQ ID NO:27-41 and derivant thereof.Preferred, PEG coupling peptide of the present invention comprises, wherein n=0 and X ¹It is the situation that contains the peptide of aminoacid shown in the SEQ ID NO:27-41 and derivant thereof.Preferred, PEG coupling peptide of the present invention can be represented by following formula:

F’-R _z’ VI

Or the last acceptable salt of its physiology, wherein:

F ' is the multivalence carrier;

R is independent under each situation to be-(X ¹)-(Y ¹) _n, wherein R is covalently bonded in F ';

X ¹And Y ¹Independent separately respectively is formula-L ¹-P ¹With-L ²-P ²Peptide;

L ¹And L ²Do not exist or independent separately for having the joint of 0-9 amino acid residue;

N is 0-3;

Z is 2-8; With

P ¹And P ²Independent separately is the bradykinin b 1 receptor peptide antagonists.

Preferred, PEG coupling peptide of the present invention comprises the PEG coupling peptide of formula VI, and wherein n is 0, and Z is 4-8, and X ¹It is the peptide that contains aminoacid shown in the SEQ ID NO:27-41 and derivant thereof.

What also be considered as a part of the present invention is the peptide conjugate with following peptide sequence, and described peptide sequence is P described herein ¹And P ²The fragment of (if existence) (being subsequence), analog or derivant, wherein, anti--B1 is active basic identical with the specifically described peptide conjugate of this paper in the external and/or body of these coupling peptides.

Term " analog " refers to the molecule of table one or more aminoacid replacement, deletion and/or increase, and it is respectively derived from formula (IV) with peptide (V), coupling peptide (not link coupled P ¹And P ²(if existence)) and/or carrier is link coupled or the amino acid whose linear array of any peptidyl joint (L) of the link coupled peptide of PEG, and these molecules and this paper specifically described similarly not coupling peptide or coupling peptide compare, anti-in its external and/or body-the B1 activity is basic identical.

According to coupling peptide analogues of the present invention usually at (P) (P ¹And/or P ²(if existence)) or (L) (L ¹And/or L ²(if existence)) one or more aminoacid replacement, deletion or insertion are arranged in the sequence.General approval conservative amino acid changes the 26S Proteasome Structure and Function that least may upset polypeptide, and the variation of approval conservative amino acid generally comprises with a structure and/or functionally similar aminoacid (for example, the similar aminoacid of side chain size, electric charge and/or shape) and replaces another aminoacid.The character of these replacements is well known in the art, and exemplary amino acid replacement is summarized in table 1 and table 2.

Table 1: aminoacid replacement

Alkalescence: Arg; Lys; His; Acid: Glu; Asp polarity: Glu; Asp; Gln; Asn; Ser; Thr hydrophilic: Asp; Glu; Asn; Ser; Thr; Tyr hydrophobicity: Ala; Met; Ile; Leu; Nor-Leu; Val aromatics: Phe; Trp; Tyr p1 amino acid: Gly; Ala; Ser; Thr; Met

Table 2: aminoacid replacement

Aminoacid	The preferred replacement	Most preferably replace
			Ala Arg Asn Asp Cys Gln Glu Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val	Gly；Leu；Ile；Asn；Pro Ala；Asn；Gln；Ser Arg；Gln；His；Lys；Ser；Tyr Asn；Ser；Thr；Gln Ala Ala；Arg；Glu；Leu；Lys；Met；Ser；Tyr Gln；Ser；Thr；Asn Asn；Gln；Lys；Tyr；Phe Tyr；Val；Met；Ala；Phe；nor-Leu nor-Leu；Ile；Val；Met；Ala；Phe Asn；Asp；Ala；Glu；Gln；Ser；Tyr Ala；Gln；Tyr；Trp；Phe Leu；Val；Ile；Ala；Met Ile；Val Ala；Asn；Asp；Gly；Lys Ala；Gly；Ile；Val；Lys Phe；Tyr；His Trp；Thr；Ser Ala；Ile；Met；Phe；Tyr；nor-Leu	Val Lys Gln Glu Ser Asn Asp Pro Arg Leu Ile Arg Leu Leu Gly Thr Ser Tyr Phe Leu

If new cysteine has still kept free sulfhydryl groups, it is preferred that A, F, H, I, L, M, P, V, W or Y are become C.

Can be in some replacement of needs determine required aminoacid replacement (conservative or nonconservative) by those skilled in the art.For example, available amino end acid replaces the affinity of identifying residue important in the peptide sequence described herein, raising or reducing not link coupled and/or coupling peptide described herein.

In some embodiments, conservative amino acid replaces also can comprise the amino acid residue that is substituted by the non-natural existence, and they normally mix with chemosynthesis.

As previously mentioned, can naturally occurring residue be classified according to common side chain character (can be used for sequence modification).For example, non-conservation replaces and can comprise that an aminoacid with a certain class replaces with an another kind of aminoacid.The residue of these replacements can be incorporated into inhuman directly in the homologous peptide of congener (orthologs) zone, or be incorporated into the not homologous zone of this molecule in.In addition, for the orientation to chain is exerted one's influence, also can modify with P or G.

When doing this modification, but the hydrophilic index of considered amino acid (hydropathic index).According to hydrophobicity and charge characteristic, each aminoacid all has been endowed a hydrophilic index, and they are: isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine/cystine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (0.4); Threonine (0.7); Serine (0.8); Tryptophan (0.9); Tyrosine (1.3); Proline (1.6); Histidine (3.2); Glutamic acid (3.5); Glutamine (3.5); Aspartic acid (3.5); Agedoite (3.5); Lysine (3.9); And arginine (4.5).

Understand the importance of aminoacid hydrophilic index in this area for the biologic activity of protein-interacting.Kyte etc., J. Mol. Biol., 157:105-131 (1982).Known some aminoacid can be had similar hydrophilic index or fractional other aminoacid and biologic activity like the reserved category still.Carry out these when changing based on hydrophilic index, the preferred hydrophilic index ± 2, especially preferred ± 1, the aminoacid replacement within preferred ± 0.5 more specifically.

Also understand in this area, can on hydrophilic basis, carry out effectively similar amino acid whose replacement.Proteic maximum local average hydrophilic (being subjected to the hydrophilic control of its contiguous aminoacid) is relevant with its immunogenicity and antigenicity, promptly relevant with this proteic biological property.

The hydrophilicity value of following amino acid residue is: arginine (+3.0); Lysine (+3.0); Aspartic acid (+3.0 ± 1); Glutamic acid (+3.0 ± 1); Serine (+0.3); Agedoite (+0.2); Glutamine (+0.2); Glycine (0); Threonine (0.4); Proline (0.5 ± 1); Alanine (0.5); Histidine (0.5); Cysteine (1.0); Methionine (1.3); Valine (1.5); Leucine (1.8); Isoleucine (1.8); Tyrosine (2.3); Phenylalanine (2.5); Tryptophan (3.4).Carrying out these based on similar hydrophilicity value when changing, the preferred hydrophilic value ± 2, especially preferred ± 1, the aminoacid replacement within preferred ± 0.5 more specifically.Also can be from the elementary Sequence Identification epi-position of aminoacid on hydrophilic basis.These zones are also referred to as " epi-position core space ".

Those skilled in the art utilize the technology know can determine the suitable analog of not link coupled and/or coupling peptide described herein.Those skilled in the art also can know and can keep its activity (replacement of conservative amino acid residue) with the residue that exists in the chemically similar aminoacid replacement native peptides.Therefore, even for biologic activity or for the very important zone of structure, also can carry out conservative amino acid and replace, and not destroy its biologic activity or not link coupled and the structure coupling peptide are not had adverse effect.

In addition, those skilled in the art can combine and look at structure-functional study, and residue very important for activity or structure is identified in these researchs in not link coupled and/or coupling peptide sequence.By relatively, can the predicted polypeptide sequence in the importance of amino acid residue.For predicting that in this way it is the amino acid residue of the not link coupled and/or coupling peptide of important amino acid whose the present invention, those skilled in the art can select with chemically similarly aminoacid replacement replace them.

Existing a lot of scientific publication things have been discussed the prediction of secondary structure specially.See Moult J., Curr.Op.inBiotech., 7 (4): 422-427 (1996), Chou etc., Biochemistry, 13 (2): 222-245 (1974); Chou etc., Biochemistry, 113 (2): 211-222 (1974); Chou etc., Adv.Enzymol.Relat.Areas Mol.Biol., 47:45-148 (1978); Chou etc., Ann.Rev.Biochem., 47:251-276and Chou etc., Biophys.J., 26:367-384 (1979).And the available computers program is assisted the prediction of secondary structure now.

Other method of prediction secondary structure comprises " threading " (" threading ") (Jones, D., Curr.Opin.Struct.Biol., 7 (3): 377-87 (1997); Sippl etc., 15-9 (1996)), " pattern analysis " (" profile analysis ") (Bowie etc., Science, 253:164-170 (1991) Structure, 4 (1):; Gribskov etc., Meth.Enzym., 183:146-159 (1990); Gribskov etc., Proc.Nat.Acad.Sci., 84 (13): 4355-8 (1987)) and " it is chain to evolve " (" evolutionary linkage ") see Home, on seeing, and Brenner, on seeing).

Can be used for same purpose (that is the active antagonist of B1 in the external and/or body) according to peptide of the present invention and/or link coupled peptide analogues and derivant and the specifically described similar peptide of this paper and/or link coupled peptide.

Peptide.Peptide of the present invention comprises: the peptide that contains sequence shown in SEQ ID NO:15-35 and the 39-54.Carrier of the present invention-or PEG coupling peptide in peptide sequence P ¹And P ²If (existence) (P) comprise as described, in conjunction with and antagonism (for example, reducing) the active peptide of B1.The preferred carrier of the present invention-or PEG coupling peptide comprise at least one peptide sequence that is selected from SEQ ID NO:5-60 and derivant thereof.Preferred, carrier of the present invention-or PEG coupling peptide comprise at least one peptide sequence that is selected from SEQ ID NO:27-41 and derivant thereof.

Table 3-Kallidin I

Receptor/effect	Peptide	Peptide sequence
			B2/B1 gaonist B2 gaonist B2 gaonist Bl gaonist Bl antagonist Bl antagonist B2 antagonist B1/B2 antagonist B2 antagonist B1 antagonist B1/B2 antagonist B1 antagonist B1 antagonist B1/B2 antagonist B1 antagonist B1 antagonist B1 antagonist B1 antagonist B1 antagonist B1 antagonist Bl antagonist B1 antagonist B1 antagonist B1 antagonist B1 antagonist B1 antagonist B1 antagonist B1 antagonist B1 antagonist B1 antagonist B1 antagonist B1 antagonist	Bradykinin; BK ( SEQ ID NO：1 ) ,Lys-BK ( SEQ ID NO：2 ) Met-Lys-BK ( SEQ ID NO：3 ) BK ( SEQ ID NO：4 ) [Leu8]-Des-Arg9-BK ( SEQ ID NO：5 ) DALK ( SEQ ID NO：6 ) ( SEQ ID NO：7 ) ( SEQ ID NO：8 ) ( SEQ ID NO：9 ) ( SEQ ID NO：10 ) ( SEQ ID NO：11 ) ( SEQ ID NO：12 ) ( SEQ ID NO：13 ) ( SEQ ID NO：14 ) ( SEQ ID NO：15 ) ( SEQ ID NO：16 ) ( SEQ ID NO：17 ) ( SEQ ID NO：18 ) ( SEQ ID NO：19 ) ( SEQ ID NO：20 ) ( SEQ lD NO：21 ) ( SEQ ID NO：22 ) ( SEQ ID NO：23 ) ( SEQ ID NO：24 ) ( SEQ ID NO：25 ) ( SEQ ID NO：26 ) ( SEQ ID NO：43 ) ( SEQ ID NO：44 ) ( SEQ ID NO：45 ) ( SEQ ID NO：46 ) ( SEQ ID NO：47 ) ( SEQ ID NO：48 )	Arg Pro Pro Gly Phe Ser Pro Phe Arg Lys Arg Pro Pro Gly Phe Ser Pro Phe Arg Met Lys Arg Pro Pro Gly Phe Ser Pro Phe Arg Arg Pro Pro Gly Phe Ser Pro Phe Arg Pro Pro Gly Phe Ser Pro Leu Lys Arg Pro Pro Gly Phe Ser Pro Leu DArg Arg Pro Hyp Gly Thi Ser DTic Oic Arg DArg Arg Pro Hyp Gly Thi Ser DTic Oic DArg Arg Pro Hyp Gly Thi Ser DHpe Oic Arg L Me- Ac ys Lys Arg Pro Pro Gly Phe Ser D-β-NaI Ile DArg Arg Pro Hyp Gly Igl Ser DIgl Oic Arg Lys Lys Arg Pro Hyp Gly Igl Ser DIgl Oic Lys Lys Arg Pro Hyp Gly Cpg Ser Dtic Cpg DArg Arg Pro Hyp Gly Igl Ser Df5f Igl Arg DOrn Lys Arg Pro Hyp Gly Cpg Ser Dtic Cpg DOtn Lys Arg Pro Thz Gly Cpg Ser Dtic Cpg 3Pal Lys Arg Pro Hyp Gly Cpg Ser Dtic Cpg 4Pal Lys Arg Pro Hyp Gly Cpg Ser Dtic Cpg Cha Arg Pro Hyp Gly Cpg Ser Dtic Cpg 2-Nal Arg Pro Hyp Gly Cpg Ser Dtic Cpg Lys Arg Pro Hyp Gly Cpg Ser Dtic Cpg DLys Lys Arg Pro Hyp Gly Cpg Ser Dtic Cpg Lys DOrn Arg Pro Hyp Gly Cpg Ser Dtic Cpg Lys Cha Arg Pro Hyp Gly Cpg Ser Dtic Cpg Lys Abu Arg Pro Hyp Gly Cpg Ser Dtic Cpg Lys 2-Nal Arg Pro Hyp Gly Cpg Ser Dtic Cpg D- Dab Lys Arg Pro Hyp Gly Cpg Ser Dtic Cpg D- Ac Dab Lys Arg Pro Hyp Gly Cpg Ser Dtic Cpg DOrn Lys Arg Pro Hyp Gly Cpg Ser Dtic Cpg Ac DOrn Lys Arg Pro Hyp Gly Cpg Ser Dtic Cpg D- 3’Pal Lys Arg Pro Hyp Gly Cpg Ser Dtic Cpg D- Ac 3’Pal Lys Arg Pro Hyp Gly Cpg Ser Dtic Cpg

B1 antagonist B1 antagonist B1 antagonist B1 antagonist B1 antagonist B1 antagonist B1 antagonist Bl antagonist B1 antagonist B1 antagonist B1 antagonist B1 antagonist

(SEQ ID NO：49) (SEQ ID NO：50) (SEQ ID NO：51) (SEQ ID NO：52) (SEQ ID NO：53) (SEQ ID NO：54) (SEQ ID NO：55) (SEQ ID NO：56) (SEQ ID NO：57) (SEQ ID NO：58) (SEQ ID NO：59) (SEQ ID NO：60)

D- D-2- Lys Nal Arg Pro Hyp Gly Cpg Ser Dtic Cpg D-2- Lys Nal Arg Pro Hyp Gly Cpg Ser Dtic Cpg D Me- Orn Arg Oic Pro Gly Phe Ser D-β-NaI Ile D Me- Ac Orn Arg Oic Pro Gly Phe Ser D-β-NaI Ile Me- DOrn Lys Arg Oic Pro Gly Phe Ser D-β-NaI Ile Ly Me- Ac DOrns Arg Oic Pro Gly Phe Ser D-β-NaI Ile Lys Arg Pro Pro Gly Phe Ser D-β-NaI Ile Ac Lys Arg Pro Pro Gly Phe Ser D-β-NaI Ile Or Me- n Arg Oic Pro Gly Phe Ser D-β-NaI Ile Or Me- Ac n Arg Oic Pro Gly Phe Ser D-β-NaI Ile Ly Me- s Arg Oic Pro Gly Phe Ser D-β-NaI Ile Ly Me- Ac s Arg Oic Pro Gly Phe Ser D-β-NaI Ile

Carrier.Term used herein " carrier " refers to prevent therapeutic peptide or protein degradation and/or increases its half-life, reduces toxicity, reduces immunogenicity or increase the molecule of its biologic activity.Can be used for carrier of the present invention and be (for example, see PCT publication WO 98/07746, include this paper in as a reference in full) known in the art and be that those skilled in the art are easy to obtain.Among the present invention, preferred carrier includes but not limited to, poly-Propylene Glycol, poly-hydroxyl propylene glycol, polypropylene glycol and oxide thereof, poly-methyl propanediol, poly-hydroxyl-expoxy propane, straight chain and side chain polypropylene glycol and derivant thereof, Polyethylene Glycol and polypropylene glycol and monomethyl ether thereof, single cetyl ether, single n-butyl ether, single tertbutyl ether and single oleyl ether, the ester of ployalkylene glycol and carboxylic acid, the dehydrating condensation product and the derivant thereof of ployalkylene glycol and amine and other polyalkylene oxides and glycol, poly-(vinylpyrrolidone), polyvinyl alcohol, poly-(ethylene acetate), copolymer poly-(ethylene acetate-be total to-vinyl alcohol), polyvinyl  oxazolidone, poly-(vinyl methyl  oxazolidone and poly-(vinyl methyl ether), poly-(acrylic acid), poly-(methacrylic acid), poly-hydroxyethyl methacrylate, poly-(acrylamide) and poly-(Methacrylamide) and its other amide, poly-(N, the N-DMAA), poly-(N-N-isopropylacrylamide), poly-(N-acetylamino acrylamide) and poly-(N-acetylamino methyl acrylamide, and other N-substitutive derivative of these amide.

An aspect of of the present present invention requires to exist at least one carrier (F) of the amino acid residue that is connected in non-peptidyl blank area or peptidyl joint, and the amino acid residue of described non-peptidyl blank area or peptidyl joint is covalently bonded in the B1 peptide antagonists.In the present invention, as described herein, preferred carrier constitutes the PEG molecule.As described herein, preferred carrier constitutes multivalence PEG molecule.

The carrier of the specifically open or indication of this paper-or the link coupled molecule of PEG can be at (X ¹)-(Y ¹) _n(as defined above) a small amount of modification is arranged in the zone, to form, as long as it has kept the antagonism to B1 substantially according to analog of the present invention.

Carrier of the present invention-or link coupled peptide of PEG and their analog between, no more than 3 of the difference of the non-terminal residue in preferred (P) district.Preferred, the analog that the present invention considers be included in carrier of the present invention-or any specific non-end site in PEG coupling peptide (P) district have two aminoacid replacement, insertion or disappearances at the most.Most preferred, carrier of the present invention-or PEG coupling peptide and their analog considered between, especially in specific (P) district, the form of difference is one or more " conservative modifications ".

Joint.Term used herein " joint " refers to L ¹And L ²If (existence) suc as formula (on seeing) shown in Iv or the V, is abbreviated as (L) herein.Preferably, be naturally occurring peptidyl (i.e. the aminoacid that links together by peptide bond) (L), form by 1-9 aminoacid.Preferred, (L) to form by 1-9 aminoacid, wherein said aminoacid is selected from 20 kinds of naturally occurring aminoacid.In preferred embodiment, the 1-9 of a described peptidyl joint aminoacid is selected from cysteine, glycine, alanine, proline, arginine, agedoite, glutamine and lysine.Preferred, the peptidyl joint is made up of uncrossed aminoacid (as glycine and alanine) on most of spaces of peptide bond connection.Therefore, preferred peptidyl joint is poly-(Gly) _1-8, especially (Gly) ₃(SEQ ID NO:100), (Gly) ₅(SEQ ID NO:101) and (Gly) ₇(SEQ ID NO:102) and poly-(Gly-Ala) _2-4With poly-(Ala) _1-8Other object lesson of peptidyl joint comprises (Gly) ₅Lys (SEQ IDNO:103) and (Gly) ₅LysArg (SEQ ID NO:104).Other combination of Gly and Ala also is preferred.In order to explain above-mentioned term, for example, (Gly) ₅Lys refers to Gly-Gly-Gly-Gly-Gly-Lys.The peptidyl joint can comprise N-end cysteine, another sulfydryl or nucleopilic reagent, to be connected with carrier.Preferred joint comprises N-end cysteine or homocysteine residue or other 2-amino-ethyl mercaptan or 3-amino-propanethiol part, so that be connected with the functionalized carrier of maleimide, iodo-acetamide or thioesters.The activatory PEG reaction of peptide and maleimide forms initial 3-sulfane base butanimide adduct (1c); with this adduct of excessive alkali treatment (1c) the butanimide adduct of less stable is converted into the 6-methyl carbamyl-5-oxo-thiomorpholine-3-Methanamide form (1d, scheme 1) with hydrolytic stability.Perhaps with the thioesters that can obtain from commercial channels or iodo acetylamino PEG (Nektar Therapeutics, Huntsville, AL) as scheme 2 with carry out chemo-selective shown in 3 and be connected.

Scheme 1

Scheme 2

Scheme 3

Another preferred joint is big, flexible joint, and it comprises Gly/Ser/Thr sequence (GSGSATGGSGSTASSGSGSATH at random; SEQ ID NO:105), it is about the PEG molecule of 1k according to estimates.In addition, the peptidyl joint can comprise non-peptidyl section, suc as formula-CH2-CH2-CH2-CH2-CH2-CH2-(rigid joint :-AEAAAKEAAAKEAAAKAGG-) 6 carbocyclic aliphatic family molecules.

Perhaps, the non-peptidyl joint that contains active nucleophilic group can be present in X ¹And Y ¹In (if existence).For example, available alkyl joint is as-NH-(CH ₂) _s-C (O)-, s=2-20 wherein.These alkyl joints can be further by the group that does not hinder on any space low alkyl group (for example, C for example ₁-C ₆) lower acyl, halogen (Cl for example, Br), CN, NH ₂, replacement such as phenyl.Exemplary non-peptidyl joint is PEG joint (as follows):

Wherein, n makes that the linkers amount is 100-5000 dalton (kD), preferred 100-500kD, m=1-3.Preferably, non-peptidyl joint is the aromatics joint.Available same method described herein changes joint to form derivant.

In addition, available PEG aldehyde carries out standard reductive alkylation or carries out acylation reaction with N-Hydroxysuccinimide or the carbonic ester of PEG, peg moiety is connected in the amine of N-terminal amine or selected side chain.Any above-mentioned joint all can be used in this method.Perhaps, suitable functionalized PEG can be directly connected in any bradykinin b 1 receptor peptide antagonists shown in SEQ ID NO:5-26 or the SEQ ID NO:43-60, or be directly connected in the amino acid residue of the peptidyl joint that is covalently bonded in any bradykinin b 1 receptor peptide antagonists shown in SEQ ID NO:5-26 or the SEQ ID NO:43-60.

Should be understood that method of the present invention may produce multiple carrier: the peptide structure because described carrier and/or target peptide may be polyvalent.For example, monovalence carrier and monovalence peptide will produce 1: 1 conjugate, and bivalence peptide and monovalence carrier can form the peptide conjugate that has two carrier parts, and bivalence carrier and monovalence peptide can produce the peptide conjugate that two peptide entities are connected in a carrier part; Employing more high price carrier can form bunch shape (clusters) peptide conjugate that a plurality of peptide entities are incorporated into single carrier, and more the high price peptide will partly be surrounded by a plurality of carriers.Peptide moiety can contain the reactive group that reacts with activatory carrier more than, must consider to form the probability of composite construction constantly.When needs form simple structure as 1: 1 the carrier and the adduct of peptide, or adopt the bivalence carrier to form peptide: carrier: during the adduct of peptide, use the activation carrier and the fret peptide of predetermined ratio, predetermined concentration, and (for example in predetermined condition, response time, temperature, pH etc.) react to form the ratio of described product down, then described product is separated with other product.Can obtain reaction condition, ratio and the concentration of reagent by simple relatively repetition test, these are tested within the limit of power of persons skilled in the art, ratio can be amplified if desired.Similarly, the purification of realizing product with routine techniques well known to those skilled in the art with separate.Yet, used as this description and claim, singulative a kind of, one, should or this (" a ", " an ", " the ") comprise a plurality of, multiple, unless context has other clear and definite regulation.Therefore, for example, " a kind of carrier-coupling peptide antagonists " or " the link coupled peptide antagonists of a kind of PEG " comprises one or more of the type Therapeutic Method that can know after the mixture that refers to these conjugates, " this Therapeutic Method " comprise that those skilled in the art are known or read this description.

Normally in being with or without the phosphate buffer of organic solvent, carry out by the PEGization that the mPEG-maleimide is connected with the sulfydryl of peptide with cysteine amino and polypeptide.The peptide of PEGization and the high dissolution of polypeptide and pyrrolidine-2,5-diketone ring possible unstability in water have hindered application and the peptide of PEGization and the application in protein purification of this method in large-scale production.Therefore, we disclose a kind of new non-aqueous condition that is used to connect mPEG-maleimide and the sulfydryl of peptide with cysteine residues and polypeptide herein.During this new method produces to the PEGization peptide and the polypeptide of high yield, and with Michael addition and amino decomposition and combination be in the same place (scheme 4).These two kinds of conditions make and can the PEGization peptide directly be separated with polypeptide by precipitation.

Scheme 4

In another embodiment of scheme 4 described methods, in conjunction with above-mentioned or following any embodiment, the available solvent of following one or more solvents that comprises replaces methanol (MeOH): methanol, ethanol, isopropyl alcohol, normal propyl alcohol, n-butyl alcohol, dichloromethane (DCM), acetonitrile (AcN), oxolane (THF), dimethyl formamide (DMF), dimethyl acetylamide (DMAc) and N-Methyl pyrrolidone (NMP).

In another embodiment of scheme 4 described methods, in conjunction with above-mentioned or following any embodiment, the available solvent of following one or more solvents that comprises replaces TBME: diethyl ether, methyl isopropyl ether and Di Iso Propyl Ether.

In another embodiment of scheme 4 described methods, in conjunction with above-mentioned or following any embodiment, scheme 4 described reactions 1) can under about 20 ℃-60 ℃ temperature, carry out.Preferably, case 4 described reactions 1) can under about 30 ℃-50 ℃ temperature, carry out.Preferred, case 4 described reactions 1) can under about 35 ℃-45 ℃ temperature, carry out.Most preferably, case 4 described reactions 1) can at room temperature carry out.

In another embodiment of scheme 4 described methods, in conjunction with above-mentioned or following any embodiment, scheme 4 described reactions 2) can under about 20 ℃-60 ℃ temperature, carry out.Preferably, case 4 described reactions 2) can under about 30 ℃-50 ℃ temperature, carry out.Preferred, case 4 described reactions 2) can under about 35 ℃-45 ℃ temperature, carry out.Most preferably, case 4 described reactions 2) can at room temperature carry out.

In another embodiment of scheme 4 described methods, in conjunction with above-mentioned or following any embodiment, scheme 4 described precipitation can be carried out 10 minutes at least.Preferred, scheme 4 described precipitation can be carried out 60 minutes at least.Most preferred, scheme 4 described precipitation were carried out 60 minutes approximately.

In another embodiment of scheme 4 described methods,, can carry out once above precipitation, filtration and/or purification reaction in conjunction with above-mentioned or following any embodiment.

In another embodiment of scheme 4 described methods,, can carry out once above precipitation and/or purification reaction in conjunction with above-mentioned or following any embodiment.

In this methods and strategies, the peptide of part protection is particularly useful, because they allow the selected sex modification of specific site of multiple functionalized peptide.From the PEG conjugate, remove blocking group with the guard method of going of having set up that the synthetic those skilled in the art of peptide know.The synthetic known orthogonally protect method of those skilled in the art of available peptide prepares the peptide of the part protection that is suitable for this purposes.Synthetic and the coupling of the bradykinin b 1 receptor peptide antagonists of part protection is illustrated in the scheme 5.The basic amino acid of the available orthogonally protect that is easy to obtain prepares the analog that side chain can be used as the coupling site.

Scheme 5

Available similar method is coupled to peg moiety with the part protection form of the B1 antagonist peptide of (SEQ ID NO:5-60) as shown in table 3.

In an embodiment of scheme 5 described methods; the amine side chain of the peptide 5c of part protection is by the protection of tert-butyl group carbamyl (Boc) part; the peptide that produces at organic solvent as 1; 2-dichloroethanes (DCE), N, in dinethylformamide (DMF) or its mixture with arbitrary reaction of above-mentioned PEG aldehyde.The formation that can add dehydrant such as Powdered 4 ° of A molecular sieves acceleration intermediate imines.After stirring at room 1-24 hour, add normal sodium triacetoxy borohydride of 1-4 or sodium cyanoborohydride with the imines reduction that produces.

In another embodiment of scheme 5 described methods,, protect the reaction of peptide such as 5c under about 20 ℃-60 ℃ temperature, to carry out with part in conjunction with above-mentioned or following any embodiment.Preferably, protect the reaction of peptide such as 5c under about 20 ℃-50 ℃ temperature, to carry out with part in the scheme 5.Preferred, protect the reaction of peptide such as 5c under about 35 ℃-45 ℃ temperature, to carry out with part in the scheme 5.Protect the great majority reaction of peptide such as 5c at room temperature to carry out with part in the scheme 5.

In another embodiment of scheme 5 described methods; the amine side chain of the peptide 5c of part protection is by the protection of tert-butyl group carbamyl (Boc) part; the peptide that produces at organic solvent as 1; 2-dichloroethanes (DCE), N, in dinethylformamide (DMF), dichloromethane, N-crassitude (NMP) or its mixture with above-mentioned PEGN-N-Hydroxysuccinimide or arbitrary reaction of p-nitrophenyl ester PEG reagent.Activatory PEG ester can be a monofunctional or linear Bifunctionalized, and these two kinds all can be by commercial sources from supplier such as Nektar or NOF acquisition.In addition, when preparation conjugate of the present invention, the activatory ester of the multiple functionalized PEG of side chain that contains 3-6 N-Hydroxysuccinimide or p-nitrophenyl ester moiety is particularly useful.

In another embodiment of scheme 5 described methods, in conjunction with above-mentioned or following any embodiment, protect the reaction of peptide such as 5c under about 20 ℃-60 ℃ temperature, to carry out with part, the response time was about 4 hours-10 days.Preferably, protect the reaction of peptide such as 5c to carry out under about 20 ℃-50 ℃ temperature with part in the scheme 5, the response time was about 12 hours-5 days.Preferred, protect the reaction of peptide such as 5c under about 35 ℃-45 ℃ temperature, to carry out with part in the scheme 5, the response time is about 1-5 days.Protect the great majority reaction of peptide such as 5c at room temperature to carry out with part in the scheme 5, the response time is about 1-4 days.

In another embodiment of scheme 5 described methods, in conjunction with above-mentioned or following any embodiment, the removal of side chain protected group can be carried out under about-20 ℃ to 60 ℃ temperature in the scheme 5.This reaction can be carried out with about 5 volume % trifluoroacetic acid (TFA)-50 volume %TFA in compatible solvent such as dichloromethane.Preferably, the removal of acid mediated blocking group can be carried out under about 0 ℃ to 40 ℃ temperature in the scheme 5, with the TFA of about 10-25 volume %.Preferred, the removal of acid mediated blocking group can be carried out under about 0 ℃ to 25 ℃ temperature in the scheme 5, and the TFA with about 10-20 volume % carries out in dichloromethane.Most preferred, the removal of acid mediated blocking group can at room temperature be carried out in the scheme 5, with the TFA of about 20 volume %, carries out in dichloromethane.

In another embodiment of scheme 5 described methods, in conjunction with above-mentioned or following any embodiment, available reversed-phase HPLC, size exclusion chromatography, ion-exchange chromatography or film dialyse the product 5d described in the purification scheme 4.Preferred, can unite or order uses above-mentioned two or more purification techniques so that the conjugate of purification of the present invention to be provided.

In another embodiment of scheme 5 described methods,, can carry out once above purification in conjunction with above-mentioned or following any embodiment.

Derivant.The present invention also considers the derivant of peptide of the present invention and/or coupling peptide.These derivants can further be improved carrier of the present invention-or solvent degree of PEG coupling peptide, absorbability, biological half life etc.Perhaps, any disadvantageous characteristic of peptide of the present invention and/or coupling peptide can be eliminated or weaken to the part of increase.Exemplary derivant comprises following carrier-or link coupled peptide of PEG, wherein:

1. described peptide and/or link coupled peptide or its some part are cyclic.For example, the peptide moiety of peptide and/or link coupled peptide can be modified to contain two or more cysteine residues (for example, in the peptidyl joint), and it can the cyclisation by forming disulfide bond.The preparation of cyclisation derivant is referring to document WO 00/24782.

2. described peptide and/or carrier-or the link coupled peptide of PEG be crosslinked or be endowed and can have carried out crosslinked characteristic intermolecular.For example, the peptide moiety of coupling peptide can be modified containing a Cys residue, thereby and can form intermolecular disulfide bond with similar molecule.

3. one or more peptidyls [C (O) NR-] connect (peptide bond) and are connected replacement by non-peptide bond.Exemplary non-peptidyl connects ₂-carbamate [CH ₂-OC (O) NR-], phosphate ester ,-CH ₂-sulfanilamide [CH ₂-S (O) ₂NR-], urea [NHC (O) NH is little-CH ₂-secondary amine and alkylation peptide [C (O) NR ⁶-, R wherein ⁶Be low alkyl group].

4.X ¹The N-terminal cysteine residues of middle coupling peptide can be replaced by the N-terminal deriveding group.Exemplary N-terminal deriveding group comprises-NHR ¹, R wherein ¹It is monoalkyl.

Derive for carrier-coupling peptide or its functional derivative is linked to water-insoluble substrate or other macromole carrier is useful with Bifunctionalized reagent.Cross-linking agent commonly used comprises; for example; 1; two (the diazonium ethanoyl)-2-diphenylphosphino ethanes of 1-, glutaraldehyde, N-hydroxy-succinamide ester, for example, with the ester of 4-azidosalicylic acid formation; with basic difunctionality imino-ester; comprise two succinimido esters as 3,3 '-dithio two (succinyl phosphorons amino propyl acid ester) and difunctionality maleimide such as two-N-dimaleoyl iminos-1, the 8-octane.Derivative reagent such as methyl-3-[(are to azidophenyl) dithio]-propionyl imino-ester (propioimidate) but produce the intermediate of photoactivation, these intermediate can form crosslinked when having light to exist.Perhaps, adopt the saccharide and the U.S. Patent No. 3,969,287 of reactive water-insoluble substrate such as cyanogen bromide-activated; 3,691,016; 4,195,128; 4,247,642; 4,229,537; With 4,330, the reactive base material described in 440 carries out protein to be fixed.

Can be connected to saccharide (oligosaccharide) group known easily be the site of the glycosylation site in the protein.Generally, the oligosaccharide that O-is connected is connected to serine (Ser) or threonine (Thr) residue, and the oligosaccharide that N-connects is connected to agedoite (Asn) residue, when this asparagine residue was Asn-Aaa-Ser/Thr sequence a part of, wherein Aaa can be any aminoacid except that proline.Aaa is one of 19 kinds of naturally occurring aminoacid except that proline preferably.The saccharide residue of finding in the structure of the oligosaccharide that the N-connection is connected with O-and each type is different.A kind of sugar that usually can find on these two kinds (the N-connection is connected with O-) oligosaccharide is N-acetyl neuraminic acid (being called sialic acid).Sialic acid normally N-connects the terminal residue of the oligosaccharide be connected with O-, owing to it has negative charge, may give glycosylated coupling peptide with acidic character.These sites can be incorporated in the joint of carrier of the present invention-coupling peptide.Available synthetic or semisynthesis known in the art is with the further glycosylation in these sites.

Other possible modification comprises the hydroxylating of proline and lysine, the phosphorylation of the oh group of seryl-or Threonyl residue, the oxidation of sulphur atom in the cysteine, (the Creighton that methylates of the alpha-amido group of lysine, arginine and/or histidine side chain, protein: structure and molecular characterization (Proteins:Structure and Molecular Properties), W.H.Freeman ﹠amp; Co., San Francisco, 79-86 page or leaf (1983)).

Except otherwise herein provided, synthesizing of peptide as herein described and/or coupling peptide, the preparation, its activation and the coupling that comprise suitable amino acid derivativges are to form the purification process and the purity testing of peptide, peptide, be included in the general knowledge scope in chemistry of peptides field, as Houben-Weyl " Methoden der OrganischenChemie " the 16th volume, I ﹠amp; The II part, (1974) described liquid phase is synthetic.For solid phase synthesis process, suitable technique also is known in the art, comprises Merrifield, Chem. polypeptide, 335-361 page or leaf (Katsoyannis and Panayotis compile) (1973); Merrifield, J.Am.Chem.Soc., the 85th volume, 2149 pages (1963); Davis etc., Biochem.Intl., the 10th volume, 394-414 page or leaf (1985); Stewart and Young, solid-phase peptide is synthesized (1969); U.S. Patent No. 3,941,763; Finn etc., protein (the 3rd edition), the 2nd volume, 105-253 page or leaf (1976); And Erickson etc., protein (the 3rd edition), the 2nd volume, the technology described in the 257-527 page or leaf (1976).Peptide synthesizes the solution technique of the chemist in field with the enough standards of energy, or synthesizes described peptide with manual or automatic solid phase method.Solid phase synthesis is the optimization technique of each peptide of preparation, and is low because it has a cost.

Pharmaceutical composition

Summation.The present invention for example also provides the method for using peptide of the present invention and/or carrier-coupling peptide in the prevention of inflammation and pain (including but not limited to inflammatory pain and relevant hyperalgesia and unusual pain) or treatment.Peptide of the present invention and/or carrier-coupling peptide pair is relevant with the B1 activation or also have therapeutic value by the prevention or the treatment of other antalgesic of its mediation, described and B1 activation is relevant or included but not limited to by other antalgesic of its mediation, the thalamic pain syndrome, diabetes, toxin and chemotherapy, septic shock, arthritis, vascular and non-vascular Combination syndrome, general inflammation, arthritis, rheumatism, lupus, osteoarthritis, inflammatory bowel, the inflammatory ophthalmic, inflammatory or unstable bladder disease, psoriasis, skin symptom with inflammatory component, sunburn, carditis, inflammatory bowel, dermatitis, myositis, the neuritis, the collagen vascular disease, the chronic inflammatory disease, epidermal tissue's damage or dysfunction, herpes simplex, diabetic neuropathy pain, postherpetic neuralgia, skin scorch pain, the sympathetic pain (sympatheticallymaintained pain) of keeping, go to import into syndrome, tension headache, angina pectoris, migraine, operation pain, breathe, Genito-urinary, the internal organs vigor disorder of gastrointestinal or angiosomes, wound, burn, allergic rhinitis, asthma, allergic skin reaction, pruritus, vitiligo, general gastroenteropathy, colitis, gastric ulcer, duodenal ulcer, vasomotion or allergic rhinitis.

The present invention also provides peptide of the present invention and/or carrier-coupling peptide in prevention or treat application in the following disease: acute headache, toothache, backache, low back pain, wound pain, operation pain, the pain that amputation or abscess cause, causalgia, demyelinating disease, trigeminal neuralgia, cancer, chronic alcoholism, apoplexy, the thalamic pain syndrome, diabetes, acquired immune deficiency syndrome (AIDS) (" AIDS "), toxin and chemotherapy, general headache, migraine, cluster headache, vascular and non-vascular Combination syndrome, tension headache, general inflammation, arthritis, rheumatism, lupus, osteoarthritis, inflammatory bowel, the inflammatory ophthalmic, inflammatory or unstable bladder disease, psoriasis, skin symptom with inflammatory component, sunburn, carditis, dermatitis, myositis, the neuritis, the collagen vascular disease, the chronic inflammatory disease, inflammatory skin and relevant hyperalgesia and allodynia, neuropathic pain and relevant hyperalgesia and allodynia, diabetic neuropathy pain, causalgia, the sympathetic pain of keeping, go to import into syndrome, asthma, allergic rhinitis, epidermal tissue's damage or dysfunction, herpes simplex, postherpetic neuralgia, breathe, Genito-urinary, the internal organs vigor disorder of gastrointestinal or angiosomes, wound, burn, allergic skin reaction, pruritus, vitiligo, general gastroenteropathy, colitis, gastric ulcer, duodenal ulcer, and bronchopathy.

Therefore, the invention still further relates to one or more peptides of the present invention and/or carrier-coupling peptide and be used for the treatment of application in the medicine of following disease for example: acute headache in preparation, toothache, backache, low back pain, wound pain, operation pain, the pain that amputation or abscess cause, causalgia, demyelinating disease, trigeminal neuralgia, cancer, chronic alcoholism, apoplexy, the thalamic pain syndrome, diabetes, acquired immune deficiency syndrome (AIDS) (" AIDS "), toxin and chemotherapy, general headache, migraine, cluster headache, vascular and non-vascular Combination syndrome, tension headache, general inflammation, arthritis, rheumatism, lupus, osteoarthritis, inflammatory bowel, the inflammatory ophthalmic, inflammatory or unstable bladder disease, psoriasis, skin symptom with inflammatory component, sunburn, carditis, dermatitis, myositis, the neuritis, the collagen vascular disease, the chronic inflammatory disease, inflammatory skin and relevant hyperalgesia and allodynia, neuropathic pain and relevant hyperalgesia and allodynia, diabetic neuropathy pain, causalgia, the sympathetic pain of keeping, go to import into syndrome, asthma, allergic rhinitis, epidermal tissue's damage or dysfunction, herpes simplex, postherpetic neuralgia, breathe, Genito-urinary, the internal organs vigor disorder of gastrointestinal or angiosomes, wound, burn, allergic skin reaction, pruritus, vitiligo, general gastroenteropathy, colitis, gastric ulcer, duodenal ulcer, and bronchopathy.

" treatment " used herein is meant the method that obtains useful or required clinical effectiveness.For purposes of the present invention, useful or required therapeutic outcome includes but not limited to following one or more: the improvement of any aspect of pain and/or inflammation or alleviation, described pain and/or inflammation comprise acute, chronic, inflammatory, nerve or postoperative pain.For purposes of the present invention, useful or required therapeutic outcome includes but not limited to following one or more: the alleviation of the alleviating of seriousness, one or more symptoms (any aspect that comprise pain and/or inflammation) relevant with pain and/or inflammation, as pain and/or the shortening of inflammation time, and/or the minimizing of pain degree or pain perception.

These pharmaceutical compositions or medicine can give by injection, oral, pulmonary, nose, transdermal or other form of medication.Generally speaking, the pharmaceutical composition that the present invention includes contains the peptide at least a of the present invention of effective dose and/or at least a carrier of the present invention-coupling peptide (its amount can effectively prevent, alleviates or eliminate pain as herein described or any other medical symptom), and pharmaceutically acceptable diluent, excipient, antiseptic, solubilizing agent, emulsifying agent, adjuvant and/or carrier.These compositionss comprise the diluent of have various buffer compositions (for example, Tris-HCl, acetate, phosphate), pH and ionic strength; Additive such as detergent and solubilizing agent are (for example, Tween 80, polysorbate 80), antioxidant (for example, ascorbic acid, sodium metabisulfite), antiseptic (for example, thimerosal (Thimerosol), benzyl alcohol) and filler (bulking substances) (for example, lactose, mannitol); These materials are incorporated into polymeric carrier-coupling peptide for example in the granular preparation of polylactic acid, polyglycolic acid etc., or mix in the liposome.Also available hyaluronic acid, it can improve the persistent period of medicine in circulation.These compositionss also can influence the interior rate of release of physical state, stability, body and the interior clearance rate of body of carrier of the present invention-coupling peptide.See, for example, Lei Mingdun pharmaceutical science (Remington ' s Pharmaceutical Sciences), the 18th edition, MackPublishing Co., Easton, PA, 1435-1712 page or leaf (1990) is included this paper in as a reference.These compositionss also can be made into liquid form, or as dry powder (for example lyophilized form).Also consider implantable slow release formulation, as preparation capable of permeating skin.

Peroral dosage form.Consider that can be used for of the present invention is oral dosage form, see that upward " Lei Mingdun pharmaceutical science " the 89th chapter is described, include this paper in as a reference.Solid dosage forms comprises tablet, capsule, pill, buccal tablet or lozenge, cachet or bolus.The encapsulated compositions of the present invention (for example, U.S. Patent No. 4,925,673 described proteinlike granules) of preparing of also available liposome or albuminoid.Available liposome methodsization, available various polymer are with liposome derivatization (see, for example, U.S. Patent No. 5,013,556).Possible solid dosage forms is described in Marshall, K., and Modern Pharmaceutics, the 10th chapter, G.S.Banker and C.T.Rhodes (1979) include this paper in as a reference.Generally, comprise carrier of the present invention-coupling peptide in the preparation, and inert fraction, these inert fraction protection carriers-coupling peptide opposing stomach environment, and carrier-coupling peptide is discharged at enteral.

What also be particularly related to is the peroral dosage form of peptide of the present invention and/or carrier-coupling peptide self.In this respect, if desired, make oral delivery effective thereby can carry out chemical modification to peptide and/or carrier-coupling peptide.Can use the salt of the aliphatic amino acid of modification, as N-(8-[2-hydroxy benzoyl] amino) sodium caprylate (SNAC), as strengthening the carrier that carrier of the present invention-coupling peptide absorbs.See U.S. Patent No. 5,792,451, exercise question is " oral drugs delivering compositions and method " (" Oral Drug Delivery Composition andMethods ").

Peptide of the present invention and/or carrier-coupling peptide can be meticulous many granules in preparation, exists with the form of about 1 millimeter size particles or bullet.The material dosage form that is used for the capsule form administration also can be a powder, or the block of mild compression (plug), or or even tablet.Can be by compression preparation therapeutic agent.

Can comprise coloring agent and flavoring agent.For example, can prepare (for example encapsulated) described peptide and/or carrier-coupling peptide or its any derivant, be included in then in the edible product, for example contain the cold drink of coloring agent and flavoring agent by liposome or microsphere.

The volume of available inert substance dilution or increase peptide of the present invention and/or carrier-coupling peptide.These diluent can comprise saccharide, especially the glucosan of mannitol, alpha-lactose, Lactis Anhydrous, cellulose, sucrose, modification and starch.Some inorganic salt comprises calcium triphosphate, magnesium carbonate and sodium chloride, also can be used as filler.Some diluent that can obtain by commercial sources are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

Can in the treatment preparation of solid dosage forms, comprise disintegrating agent.Material as disintegrating agent includes but not limited to that starch comprises the commercial obtainable disintegrating agent Explotab based on starch.Also available Sodium Carboxymethyl Starch, Amberlite, sodium carboxy methyl cellulose, ultramylopectin, sodium alginate, gelatin, Pericarpium Citri junoris, carboxylic methyl cellulose, natural sponge and bentonite.Other form of disintegrating agent is insoluble cation exchange resin.Powdered natural gum also can be used as disintegrating agent and binding agent, can comprise Powdered natural gum such as agar, karaya or tragacanth.Alginic acid and sodium salt thereof also are useful disintegrating agents.

Also useful binders keeps together each component in the pharmaceutical composition to form a hard tablet, and binding agent comprises the material from natural prodcuts, as arabic gum, tragacanth, starch and gelatin.Other binding agent comprises methylcellulose (MC), ethyl cellulose (EC) and carboxy methyl cellulose (CMC).Polyvinyl pyrrolidone (PVP) and hydroxypropyl emthylcellulose (HPMC) also can be used on and make the therapeutic agent granulating in the alcoholic solution.

Can comprise also in the preparation that friction resistant reagent bonds in the process for preparation preventing.Layer between lubricant useful as therapeutics and the die wall, lubricant include but not limited to, stearic acid comprises its magnesium salt and calcium salt, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oil and wax.Also available soluble lubricant, as sodium lauryl sulphate, Stepanol MG, various molecular weight polyethylene glycol, Carbowax 4000 and 6000.

Can add the flow behavior that to improve carrier in the process for preparation-coupling peptide and in compression process, assist the fluidizer of resetting.These fluidizer can comprise starch, Pulvis Talci, pyrogenic silica and hydrated aluminosilicate.

In order to help peptide of the present invention and/or carrier-coupling peptide to be dissolved in the aqueous environments, can add surfactant as wetting agent.This class surfactant comprises anionic detergent such as sodium lauryl sulphate, dioctyl sodium sulfosuccinate and sodium cetanesulfonate.Available cationic detergent, this cationoid detergent can comprise benzalkonium chloride or Benzene Chloride ethamine.Can be included in that the possible nonionic detergent as surfactant is Polidocanol, Polyethylene Glycol 40 stearate, polyoxyethylene hydrogenated Oleum Ricini 10,50 and 60, glyceryl monostearate, polysorbate 40 in the preparation, 60,65 and 80, sucrose fatty acid ester, methylcellulose and carboxymethyl cellulose.These surfactants separately exist in the preparation or mixture in varing proportions is present in the preparation.

Also can comprise additive in the preparation to strengthen the absorption of described peptide and/or carrier-coupling peptide.The additive that may have this specific character comprises various fatty acids, for example, and oleic acid, linoleic acid plus linolenic acid.

May need controlled slow release formulation.Peptide of the present invention and/or carrier-coupling peptide can be incorporated into the inert base that allows to discharge by diffusion or leaching mechanism medicine, for example natural gum.Also can be with the substrate of slowly degenerating, for example alginate or polysaccharide are incorporated in the preparation.The another kind of form of controllable sustained-release dosage form of the present invention is to use the method based on Oros therapy system (Alza Corp.) to prepare, and promptly medicine is surrounded by semipermeable membrane, because osmotic effect, this semipermeable membrane allows water to pass through, and promotes medicine and discharges by a little perforate.Some enteric coatings also have slowly releasing effect.

The pulmonary delivery form.This paper also considers the pulmonary delivery of pharmaceutical composition of the present invention.Described peptide and/or carrier when air-breathing-coupling peptide (or derivatives thereof) is delivered to mammal pulmonary, passes lung internal layer epidermis in blood flow.This respect may comprise Adjei etc. by the helpful report relevant with macromolecular pulmonary delivery, Pharma.Res., the 7th volume, 565-569 page or leaf (1990); Adjei etc., Internatl.J.Pharmaceutics, the 63rd volume, 135-144 page or leaf (1990) (leuprorelin acetate); Braquet etc., J.Cardiovasc.Pharmacol., the 13rd volume (supplementary issue 5), 143-146 (1989) (endothelin-1); Hubbard etc., Annals Int.Med., the 3rd volume, 206-12 page or leaf (1989) (alpha1-antitrypsin); Smith etc., J.Clin.Invest., the 84th volume, 1145-1146 page or leaf (1989) (α 1-protease); Oswein etc., proteinic aerosolization (" Aerosolization ofProteins "), Proc.Symp.Resp.Drug Delivery II, Keystone, Colorado (1990) (recombinant human somatropin); Debs etc., J.Immunol., the 140th volume, 3482-3488 rolls up (1988) (interferon-and tumor necrosis factor); And U.S. Patent No. 5,284,656 (granulocyte colony-stimulating factor).

Consider to can be used for to be the machinery that is designed for the therapeutic products pulmonary delivery, to include but not limited to of the present invention, aerosol apparatus, metered dose inhaler, powder inhalator, those skilled in the art are very familiar for these.Some object lessons that are suitable for use in commercially available device of the present invention are Mallinckrodt, Inc., St.Louis, the Ultravent aerosol apparatus that Missouri produces; Marquest Medical Products, Englewood, the Acorn II aerosol apparatus that Colorado produces; Glaxo Inc., Research Triangle Park, the Ventolin metered dose inhaler that North Carolina produces; And Fisons Corp., Bedford, the Spinhaler powder inhalator that Massachusetts produces.

All these devices all require to use and are suitable for disperseing the preparation of peptide as herein described and/or carrier-coupling peptide and/or its derivant.Usually, each preparation is narrow spectrum for the device of used a certain type, useful diluent, excipient adjuvant and/or the carrier, may also comprise and use suitable propellant material in using treatment.

The pharmaceutically acceptable carrier that is used for pulmonary's compositions comprises sugar, as trehalose, mannitol, xylitol, sucrose, lactose and sorbitol.Other composition that is used for preparation comprises DPPC, DOPE, DSPC and DOPC.Can use natural or synthetic surfactant.Can use PEG (or even use separately with the derivatization of peptide).Also glucosan can for example be used in buffer preparation, as cyclodextrin, bile salts, cellulose and cellulose derivative.Also can use aminoacid, for example in buffer preparation.

In addition, consider to use liposome, microcapsule or microsphere, the carrier of inclusion complexs or other type.

Be suitable for the preparation with aerosol apparatus (Puffer type or ultrasonic type) use, peptide as herein described that it comprised usually and/or carrier-coupling peptide is water-soluble with the concentration of about 0.1-25 milligram (mg) biological active agents/ml soln.Said preparation also can comprise buffer agent and simple sugar (for example, in order to make stabilized peptide and control osmotic pressure).Spray agent also can contain surfactant, to reduce or to prevent because the protein aggregation of the spatial induction that the atomizing of solution causes in the aerocolloidal process of formation.

The preparation that uses with the metered dose suction apparatus generally comprises the powder of segmentation, peptide of the present invention that these powder contain and/or carrier-coupling peptide at the auxiliary low suspension of surfactant in propellant.Propellant can be any conventional material that is used for this purpose, for example chlorofluorocarbon, hydrochlorofluorocarbon, and hydrogenation fluorohydrocarbon or Hydrocarbon comprise Arcton 11, dichlorodifluoromethane, dichloro-tetrafluoro ethanol and 1,1,1,2-tetrafluoroethane, and compositions.Suitable surfactant comprises sorbitan trioleate and soybean lecithin.Oleic acid is useful as surfactants also.

The dry powder that comprises segmentation with the dispersive preparation of powder inhalation device, these dry powder contain peptide of the present invention and/or carrier-coupling peptide, also can comprise filler, as lactose, sorbitol, sucrose, mannitol, trehalose or xylitol, the amount of filler helps powder to disperse from device, for example is the 50-90 weight % of preparation.

The nose delivery form.Consider that also the nose of described peptide and/or carrier-coupling peptide sends.Nose is sent and is made after giving nose with therapeutic products peptide of the present invention and/or carrier-coupling peptide directly enter blood flow, and product needn't be in pulmonary deposition.The preparation that is used to send comprises the preparation of preparing with glucosan or cyclodextrin.Also consider to send by passing other adhesive film.

Pumping.In some embodiments of the present invention, consider that local delivery peptide of the present invention and/or coupling peptide are used for the treatment of or prevent the disease of B1 mediation.The present invention considers that a kind of method of local delivery is the localized site injectable drug of having an effect at medicine.

The another kind of method of local delivery comprises and will carry the conduit of medicine to be inserted into required health site, promotes medicine with pump and pass through conduit.Outside deterioration (worn) drug efflux pump that uses with inner implantation catheter is well known to those skilled in the art.

The another kind of method that is used for local delivery comprises implantable drug delivery device.Developed the technical disadvantages that implantable pump has solved use external pump well known to those skilled in the art and conduit system.Implantable drug delivery pump often comprises: bin is used for storage of pharmaceutical; Injection port, the fresh pharmaceutical preparation of injectable and at interval old medicine is removed from bin with certain hour; And optional conduit, be used to deliver a medicament required place.Preferred implantable device includes but not limited to, the Duros implantation device (AlzaCorporation, Mountain View, CA), SynchroMed I or II filling system (Medtronic, Inc., Minneapolis) etc.

(perhaps) in addition the invention provides and be used for the above-mentioned and/or well known by persons skilled in the art any slow release of this paper or hold release dosage form or the peptide of particulate formulations and/or link coupled peptide.

Dosage.Can measure the peptide of the present invention that gives and/or the effective dose of coupling peptide by method well known in the art, but the parameter that these methods are considered comprises biological half life, biology availability and toxicity.In a preferred embodiment, those skilled in the art use the data that obtain the research in the external and body of routine well known to those skilled in the art to determine effective dosage ranges.For example, the cell in vitro culture experiment, as following embodiment 6 described exemplary tests, the data that those skilled in the art provide according to this test, can determine at an easy rate to suppress the mean inhibitory concentration (IC) of required peptide of the inductive activity of a certain amount of B1 or coupling peptide or average effective concentration (EC) (for example 50%, IC ₅₀Or 90%, IC ₉₀).Those skilled in the art use the pharmacokinetic data (embodiment 9 described pharmacokinetic datas for example) that obtains from one or more conventional animal models then, can select proper dosage, thereby obtain identical or above the peptide minimum plasma concentration (C of this value with the IC value of determining _Min).Being used for the treatment of the dosage that comprises in certain disease or the treatment of conditions method will be decided by the treatment doctor, he will consider to influence the various factors of healing potion effect, as patient's age, situation, body weight, sex and diet, the order of severity of disease to be treated, administration time, and other clinical factor.Usually, the daily dose scheme should be in 1.0-10000 microgram (μ g) peptide and/or carrier-coupling peptide/kilogram (kg) body weight, preferred 1.0-1000 μ g/ kg body weight, most preferably 1.0-150 μ g/ kg body weight.

Therapeutic alliance.On the other hand, the present invention includes treatment (or being prevention in other embodiments) pain and/or inflammation or activate the relevant any disease or the method for disease with B1, this method comprises: give a certain amount of peptide of the present invention and/or coupling peptide and a certain amount of NSAID.Term " NSAID " refers to nonsteroidal anti-inflammatory compound.Relative consumption and the ratio of peptide antagonists and/or link coupled peptide antagonists and NSAID can change.In some embodiment, give enough peptides (one or more) and/or coupling peptide (one or more), thereby feasible equal pain or the inflammation of realizing alleviated the normal dose of required NSAID and can be reduced.In some embodiment, give enough peptides of the present invention (one or more) and/or coupling peptide (one or more), thereby feasible equal pain or the inflammation of realizing alleviated the normal dose of required NSAID and can be reduced, described pain or inflammation are alleviated at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, be at least about 50%, be at least about 60%, be at least about 70%, be at least about 80%, or at least about 90%, or more.On the dosage when minimizing that the pain that described realization is equal or inflammation are alleviated the normal dose of required NSAID can be reflected in certain administration, and/or on the dosage in the preset time section (administration frequency minimizing).

On the other hand, the invention provides and strengthen the method for NSAID to the treatment of pain or inflammation, this method comprises unites the NSAID that gives effective dose and the peptide at least a of the present invention and/or at least a coupling peptide of the present invention of effective dose." unite and give " used herein (or " administering drug combinations ") comprises that NSAID and peptide of the present invention and/or coupling peptide give individual used situation with effective dose." administering drug combinations " used herein comprises administration simultaneously and/or at different time administrations.Administering drug combinations also comprises as same preparation (that is, there be (combination) in peptide of the present invention and/or coupling peptide and NSAID in same compositions) administration, and/or as the compositions administration that separates.Should be understood that administration frequency and/or interval administration that peptide of the present invention (one or more) and/or coupling peptide (one or more) and at least a NSAID can be different.For example, coupling peptide of the present invention can be administered once weekly, and the administration that NSAID can be more frequent.Should be understood that available identical or different route of administration gives peptide of the present invention (one or more) and/or coupling peptide (one or more) and NSAID, becomes different dosage regimens in the administration cycle.Even can administration before pain or inflammation begin.Therefore, on the other hand, the invention provides the treatment patient pain and/or inflammation, reduce its sickness rate, alleviation and/or postpone the method for its generation or development, described method comprises at least a NSAID that unites at least a peptide of the present invention who gives effective dose and/or at least a coupling peptide and effective dose.These methods comprise treatment or prevent any pain and/or the inflammation of any cause of disease, comprise giving pain and/or the inflammation that NSAID treats usually.These methods also are suitable for treatment or prevent this paper above-mentioned or following by B1 activation and mediation or relative any disease or disease.In some embodiment, described pain and/or inflammation are postoperative pains.In some embodiment, described pain and/or inflammation are relevant with burn or wound.In some embodiment, described pain and/or inflammation are relevant with rheumatoid arthritis.In some embodiment, described pain and/or inflammation are relevant with osteoarthritis.In some embodiment, described pain and/or inflammation are relevant with postherpetic neuralgia.In some embodiment, NSAID is selected from aspirin, acetaminophen, ibuprofen, indometacin, naproxen, naproxen, diclofenac, ketone ibuprofen, Tolmetin, sulindac, mefenamic acid, meclofenamic acid, Diflonid, Rufenisal, piroximone, sudoxicam, isoxicam, celecoxib, rofecoxib, DUP-697, flosulide, meloxicam, 6 methoxyl groups-2 naphthyl acetic acid, MK-966, Nabumetone, nimesulide, NS-398, SC-5766, SC58215, T-614, or these mixture.

Embodiment

The following example just is used for illustrative purpose, is not or should not be construed as and limit the present invention by any way.It will be understood by those skilled in the art that and to modify and change and do not break away from the spirit or scope of the present invention chemical compound described herein.Can synthesize chemical compound of the present invention according to one or more following methods.It should be noted that to show conventional method to have the preparation that does not indicate stereochemical chemical compound because this relates to.Yet these methods generally can be used for having specific stereochemical chemical compound, and for example, the spatial chemistry of certain group is (S) or (R).In addition, utilize the method for knowing, for example by reversing (inversion), Chang Keyong has a kind of spatial chemistry, and (the normal preparation of the chemical compound of (for example, (R)) has the chemical compound of opposite spatial chemistry (that is, (S)).

Synthetic and the purification of embodiment 1:B1 receptor peptide antagonists and the link coupled B1 receptor of PEG peptide antagonists

Synthesize various peptide of the present invention with synthetic technology well known in the art.The method for optimizing of the synthetic various peptides of the present invention adopts the FMOC method to utilize the activation of carbodiimides, and is as described below.

Part 1: be added on the resin with carbodiimides chemical method dissolving Fmoc-aminoacid.

Fmoc-aminoacid (3-4 equivalent) is dissolved in the dried DCM/NMP mixture (NMP or DMF with help dissolve fully).The N-hydroxybenzotriazole (HOBt, identical with the aminoacid equivalent) that will be dissolved in NMP joins in the Freamine.Be dissolved in the N of DCM, N '-dicyclohexyl-carbodiimides (DCC, identical with the aminoacid equivalent) joins in the Freamine.Mixed this solution about 20 minutes.Then activatory acid solution is added to (disgorging before being added on the resin if desired) on the resin.Stirring this reaction, to detect resin up to 1,2,3-indantrione monohydrate test negative.After coupling finishes, collect resin, several times with the DMF washing.

Part 2: remove N-end Fmoc from peptide-resin.

Handle the peptide-based resin 3 minutes of Fmoc-protection with piperidines/DMF (2/8).Drain (drain) resin, this is handled and continues 15 minutes.Use the DMF washing resin, then for several times with the DCM washing.If next step comprises as described in step 3 peptide is ruptured from resin, then this moment is air-dry with resin.

The 3rd part: TFA ruptures and goes protection.

The resin of drying in the step 2 is placed flask, add 10-25ml fracture liquid (95%TFA, 2.5% water, 1.5% tri isopropyl silane and 1% dithioglycol)/g resin.Stirred this reaction 3-4 hour, filtration under diminished pressure is removed resin, uses the TFA washed twice.Decompression down with rotary evaporation filtrate is concentrated to～20%.Cool off this liquid to-50 ℃, with the cold dry diethyl ether precipitation of 10 times of volumes.The collecting precipitation thing.Then peptide is dissolved in the water/acetonitrile mixture that contains 0.5%TFA lyophilizing.Use C18HPLC then, gradient is 10% acetonitrile/0.1%TFA aqueous solution-50% acetonitrile/0.1%TFA aqueous solution, the purification crude product.For 1 gram crude product, on Agilentprep HPLC, use 250 * 50mm C18 post, flow velocity is 90 ml/min, 215 and 254nm detect two kinds of wavelength.With the injection classification, then with each fraction of mass spectral analysis.According to mass spectrum each is managed enrichment, concentrating under reduced pressure is to remove acetonitrile, and lyophilizing obtains the B1 peptide antagonists of white powder.Characterize with HPLC-MS and Maldi-TOF mass dete ctor (mass determination).

The link coupled peptide of the various PEG of preparation the present invention as described below

The synthetic various active bradykinin b 1 receptor peptide antagonists that is selected from SEQ ID NO:5-60 has different peptidyl joints at its N-terminal, makes the terminal penultimate of each peptide antagonists contain cysteine with preceding method (for example SEQ ID NO:27-41).Use-case such as following method A or method B by the sulfydryl of the N-terminal cysteine of these peptide analogues of fixing a point the activatory polymer of maleimide is coupled to, carry out derivatization with different Polyethylene Glycol (PEG) big or small and configuration with peptide analogues.With the PEG-peptide conjugate that the ion-exchange chromatography purification produces, lyophilizing concentrates or diafiltration concentrates, and before the biologic test it is carried out dialysis in the buffer in vitro and in vivo.

Method A:

At 50mM NaHPO ₄, among 5mM EDTA, the pH 6.5, will contain the peptide and the PEG-maleimide (maleimide of excessive 1.2 times of moles on 2.5-5mg/ml peptide and the reactive chemistry meterological: reaction mercaptan), the link coupled peptide of preparation PEG of cysteine.Room temperature (20-25 ℃) stirred this reaction 1-1.5 hour.Stir one and finish, just with the beta-mercaptoethanol of 10 times of molar excess (β-ME): maleimide cessation reaction, room temperature restir 30-60 minute.

Inject 5 μ l reactant liquor to 4.6 * 250mm, 5 microns C4 post (Grace Vydac, Columbia, MD; Article number: #214TP54), with reversed-phase HPLC (RP-HPLC) detection reaction process.With the unreacted peptide of 5-90% acetonitrile linear gradient elution and the PEG-peptide conjugate that are dissolved in 0.1% trifluoroacetic acid.Usually, consumed in the reaction＞90% peptide analogues.

(MW=5kD or 20kD, PD=1.01-1.02) by Shearwater Corp. or NOF Corp, (Toyko Japan) provides the activatory PEG polymer of linear maleimide.

Purification:

In cation-exchange chromatography, use 10mM NaOAc, 20%EtOH, the link coupled peptide of SP SepharoseHP post (Amersham Biosciences) purification PEG of pH 4 pre-equilibrations.Before the application of sample,, and pH is adjusted to 3.5 with glacial acetic acid with 10 times 20% ethanol dilution reactant mixture.Diluted reactant mixture is added on the post of suitable size peptide: the ratio of resin is no more than 2.5mg/ml.

Use (CVs) 10mM NaOAc of twice column volume then, 20%EtOH, pH 4 these posts of washing are with the 10mM NaOAc that is dissolved in 10-20CV, 20%EtOH, the 0-200mM NaCl linear gradient elution among the pH 4.The absorbance of monitoring 254nm or 220nm detects the peptide and the PEG-peptide conjugate of unmodified.Under these conditions, excessive PEG and β-ME are washed out in the unconjugated outflow component, and conjugate is eluting in the broad peak that about 50mM NaCl rises, and free peptide is split well, and it is eluting when about 200mM NaCl.

The peak value component of eluting is assessed with RP-HPLC, carries out enrichment according to holdup time consistent with the PEG-peptide conjugate and homogeneity.The peak value conjugate of dry enrichment method, water is rebuild then, buffer dialysis relatively.Perhaps, available diafiltration concentrates, and conjugate is carried out buffer-exchanged.

Analyze final PEG-peptide conjugate with RP-HPLC and merge thing, usually～the 98%th, conjugate.Measure the composition and the concentration of conjugate with amino acid analysis, peptide sequencing and extinction spectrum.

With above-mentioned CEX method (Fig. 1 (A)), in the phosphate buffered saline (PBS) (PBS) of pH=7.2, monitor the stability of solution of chemical compound shown in the 1c in the interior scheme 1 of a period of time under the room temperature.The result shows chemical compound 1c because the hydrolysis of butanimide part is converted into 1d and other two kinds of products (with the structure of IR, MS/MS and NMR test simultaneous determination) very soon.

Method B:

Mechanical agitator, hygrosensor and N are being housed ₂In the 3 neck round-bottomed flasks of import, mPEG-maleimide (1.0 equivalent) is dissolved among the anhydrous MeOH under 30 ℃.After the mPEG-maleimide dissolved fully, the peptide (1.3eq.) that will contain the N-terminal cysteine residues joined in the clear solutions, stirring at room 3 hours.Reversed-phase HPLC shows that the mPEG-maleimide disappears, and the new peak of initial 3-sulfane base-butanimide adduct occurred.Next step, (Sigma-Aldrich Corp., St.Louis MO) join in the solution 10 normal diisopropyl-ethamine, and 25 ℃ were stirred 24 hours at least.In ion-exchange chromatography, as immobile phase, monitor this reaction with TOSOHAAS SP-5PW (20 μ m).CEX analyzes demonstration, and conversion ratio surpasses 98%, and remaining 3-sulfane base-butanimide adduct is less than 1.5%.Add tert-butyl group MEE (TBME) (volume is the twice of used methanol in the reaction), the turbid liquid stirring at room of generation 2 hours.Elimination white depositions, room temperature vacuum drying are spent the night to produce rough 6-methyl-carbamyl-5-oxo-thiomorpholine-3-carbamyl cross-linking products (1d).

Purification:

With RP-HPLC with MeOH-H ₂O-AcOH system (c18YMC ODS NQ is as immobile phase) the above-mentioned crude product of purification is to produce the 6-methyl-carbamyl-5-oxo-thiomorpholine-3-carbamyl cross-linking products of reversed phase chromatography mensuration purity＞98% by analysis.The component of purification is collected in together, is concentrated into drying under the vacuum, the white residue that produces is dissolved among the hot MeOH (～30 ℃) of the minimum of enough generation settled solutions, use TBME (volume is the twice of used MeOH) to handle then.The turbid solution stirring at room that produces 1 hour, elimination precipitate, room temperature vacuum drying at least 16 hours.Obtain cream-coloured pure products (1d), gross production rate is 74%,＞98%CEX and RPC purity.Measure the composition and the peptide content of conjugate with the combination of amino acid analysis, peptide sequencing, multinuclear NMR method and extinction spectrum.With above-mentioned CEX method, room temperature is monitored the stability of solution of chemical compound 1d in a period of time in the phosphate buffered saline (PBS) (PBS) of pH=7.The stability that proves this chemical compound is significantly higher, does not observe significant change in 6 days.

Analyze anti-phase (RP) and cation (CEX) chromatography in the Agilent 1100HPLC system that has diode array or variable-wavelenght detector and the automatic injector of constant temperature.The sign chromatography condition is as follows.

1.RP-HPLC the condition post of method: YMC ODS-AQ, 3 μ m, 120 , 4.6 * 100mm

Column temperature .40 ℃

Mobile phase: A) water-soluble 0.1%TFA

B) be dissolved in the 0.1%TFA of MeOH

Flow velocity: 1.1 ml/min

Gradient: time %B

0 5

10 40

30 95

35 95

35.1 5

40 5

Detect: UV, 220nm

Inject volume: 20L or 50L, depend on sample concentration

Sample concentration: 2.5-10mg/mL

Sample diluting liquid: Dulbecco ' s PBS and other buffer that is used for stability study

2. the cationic layer analysis method of analyzing commonly used

Post: TOSOH, TSK-GEL, SP-5PW, 10 μ m, 7.5 * 75mm

25 ℃ of post concentration

Mobile phase: A) the 20mM NaH among water-soluble/EtOH (8: 2) ₂PO ₄, pH ' 3.5

B) the 20mM NaH among water-soluble/EtOH (8: 2) ₂PO ₄With 0.5M NaCl, pH 3.5

Flow velocity: 1.0 ml/min

Gradient: time B%

0 0

3 0

25 45

40 100

45 100

45.1 0

50 0

Detect: UV, 220nm

Inject volume: 10-50 μ L, depend on sample concentration

Sample concentration: 2.5-10mg/mL

Sample diluting liquid: Dulbecco ' s PBS and other buffer that is used for stability study

Embodiment 2: the synthetic and purification that carries out the link coupled B1 receptor of PEG peptide antagonists with the PEG thioesters.

At 50mM NaHPO ₄, among 5mM EDTA, the pH7, will contain the peptide and the PEG-maleimide (maleimide of excessive 1.2 times of moles on 2.5-5mg/ml peptide and the reactive chemistry meterological: reaction mercaptan), the link coupled peptide of preparation PEG of cysteine.Room temperature (20-25 ℃) stirred this reaction 18-26 hour.Stir one and finish, just with the beta-mercaptoethanol of 10 times of molar excess (β-ME): maleimide cessation reaction, room temperature restir 30-60 minute.With this reactant mixture of method A purification described in the foregoing description.

Embodiment 3: the synthetic and purification that carries out the link coupled B1 receptor of PEG peptide antagonists with PEG thioesters or iodo acetas.

At 50mM NaHPO ₄, among 5mM EDTA, the pH7, will contain the peptide and PEG-OPTE (adjacent pyridine radicals thioesters) (the activatory PEG of excessive 1.2 times of moles on 2.5-5mg/ml peptide and the reactive chemistry meterological: reaction peptide), the link coupled peptide of preparation PEG of N-terminal cysteine.Room temperature (20-25 ℃) stirred this reaction 18-26 hour.Stir one and finish, just with the cysteine of 10 times of molar excess: excessive PEG reagent cessation reaction, room temperature restir 30-60 minute.With this reactant of method A purification described in the foregoing description 1.

Perhaps, can form conjugate with the PEG-iodoacetamide as mentioned above, wherein peg moiety connects (scheme 3) by thioether bond.In this case, use the activated PEG reactant of 1.5 molar equivalents, the response time is increased to 24 hours, with the β mercaptoethanol cessation reaction of w/10 molar equivalent, carries out purification as described in above-mentioned embodiment.

Embodiment 4: the synthetic and purification that carries out the link coupled B1 receptor of PEG peptide antagonists with the PEG propionic aldehyde.

The described method of available United States Patent (USP) 5,824,784 (include this paper as a reference in full) is with the N-terminal of PEG selective modification B1 receptor peptide antagonists as peptide antagonists shown in SEQ ID NO:5-60 and 27-41 are arbitrary.For example, (245mg 0.14mmol) is dissolved in 10mL and contains 100mMNaH with the peptide shown in the SEQ ID NO:6 ₂PO ₄With 60mM NaCNBH ₃Solution in.Fully stirring is cooled to 4 ℃ with this mixture, and (Nektar Therapuetics, Hu ntsville AL) handle with 2.35 gram 20K mPEG propionic aldehyde.Stir this mixture 3 days, and as described in embodiment 1 method B, used RP and CEX chromatography purification then.

Perhaps, according to the method shown in the scheme 5, the PEG that contains the amine reactive functional groups can react with the B1 peptide antagonists of part protection.After the coupling reaction, the method for knowing with solid phase and the synthetic those skilled in the art of liquid phase peptide cuts off the side chain protected group, as mentioned above the PEG-peptide construction of purification generation.Multifunctional PEG aldehyde (3-6 reactive group) also can with the protection reactive polypeptide of molar excess, produce multivalence PEG construction, wherein a plurality of peptides are to be connected according to the mode of determining on chemical regions and the stoichiometry.

Embodiment 5: the synthetic and purification that carries out the link coupled B1 receptor of PEG peptide antagonists with PEG N hydroxyl-butanimide.

According to the method shown in the scheme 5, the B1 peptide antagonists of available part protection carries out selectivity PEGization on the specific N-terminal of the arbitrary B1 receptor peptide antagonists shown in SEQ ID NO:5-60 or side chain nitrogen.For example; with part (protection) decapeptide (1.43g; 1.025mmol) solution and 3.5g (0.18mmol) Sunbright PTE-200GS (20kD 4-arm succinimido glutarate (gluterate) in the 2.5ml dry DMF; NOF; Tokyo Japan) mixes with the 1.0mL diisopropylethylamine that is dissolved in the 25mL dichloromethane.The colourless solution stirring at room that produces 2 days, reduction vaporization then.The residue that produces is dissolved in the 25mL deionized water, places 10, the dialyzer of 000MW cutoff value (Pierce, Rockford Il, USA) in.Chemical compound is to 24 hours (changing 3 times buffer) of water dialysis, and lyophilizing is to produce the tetravalence PEG product of protection then.The white solid that produces is dissolved in the 60mL dichloromethane, handles with the anhydrous TFA of 20mL.Stirring at room two days later, this reactant mixture of reduction vaporization dissolves as mentioned above then and dialyses.The material lyophilizing of dialysing is carried out purification with ion-exchange chromatography then as mentioned above, produces the tetravalence product of white solid.In a similar approach, can to adopt the PEG that contains 1-6 succinimido glutarate (gluterate) to prepare single-or polyfunctional peptide construction.

Table 4a:X ¹Peptide

SEQ ID NO：	X 1The sequence of peptide
		27	{N}CGGGKRPPGFSPL{C}
28	{N}CGGGGGKRPPGFSPL{C}
		29	{N}CGGGGGKKRPGFSPL{C}
30	{N}CGGGGGKRKRPPGFSPL{C}
		31	{N}CG-CH2-CH2-CH2-CH2-CH2-CH2-KRPPGFSPL{C}

32	{N}CGGGGGKKRPPG[AMeF]S[D-β-Nal]I{C}
		33	{N}CGGGGGKKRP[Hyp]G[Cpg]S[DTic][Cpg]{C}
34	{N}CGGGGGGGKKRP[Hyp]G[Cpg]S[DTic][CPG]{C}
		35	{N}ac-CGGGGGKKRP[Hyp]G[Cpg]S[DTic][Cpg]{C}
36	{N}KKRP[Hyp]G[Cpg]S[DTic][Cpg]{C}
		37	N} acyl group-KKRP[Hyp] and G[Cpg] S[DTic] [Cpg] { C}
38	{N}CKRPPGFSPL{C}
		39	{N}CGGGGG[DOrn]KRP[Hyp]G[Cpg]S[DTic][Cpg]{C}
40	{N}CGGGGG[DOrn]KRP[Thz]G[Cpg]S[DTic][Cpg]{C}
		41	{N}CGGGGGK[DOrn]RP[Hyp]G[Cpg]S[DTic][Cpg]{C}

Table 4b:Y ¹Peptide

SEQ ID NO：	Y 1The sequence of peptide
		42	{N}GGGGGKKRPPGFSPL{C}

The external B1 of the active peptide of embodiment 6:B1/PEG coupling peptide antagonists suppresses activity test

With following A, B and the described test of C, the peptide and/or the link coupled peptide that can selectivity suppress B1 activity (with respect to the B2 activity) have been identified.

A. use calcium flux (Calcium Flux) to say the in vitro tests of capable people B1 function of receptors:

The activation of the B1 receptor of Gq association causes the increase of intracellular Ca2+.Therefore, calcium sensitive luminescent protein (aequorin) can be used as the indicator of B1 receptor activation.Aequorin is the luminescent protein of a 21-kDa, forms the bioluminescence complex when it is connected in chromophore cofactor coelenterazine (coelenterazine).Calcium is with after this complex combines, and the oxidation reaction of coelenterazine causes apoenzyme aequorin (apoaequorin), amide coelenterazine (coelenteramide), CO ₂And the generation of light (it can be detected by the luminous detection instrument of routine).

Set up stable CHO D-/people B1 receptor (GenBank accession number AJ238044)/aequorin cell line, these cells maintain in revolving bottle in the suspension, and this suspension contains DMEM and HAM F12 (Gibco 11765-047), high glucose (Gibco 11965-084), 10% hot deactivation dialysis serum (Gibco 26300-061), 1X non essential amino acid (Gibco 11140-050), 1X glutamine-Pen-Strep (Gibco 10378-016) and the hygromycin 300 μ g/ml (Roche 843555) of 1: 1 ratio.Before luminometer test 15-24 hour, 25,000 cells/well (2.5E6 cell/10 milliliter/plate) are inoculated into the bottom transparent side surfaces are in the 96 hole bread boards of black (Costar#3904).

Culture medium is removed from the hole, changed, wherein be added with 30mM HEPES (pH7.5) and 15 μ M coelenterazine (coelenterazine h Luciferin#90608 not contain 60 μ l HAM ' s F12 of serum; AssayDesigns (Ann Arbor, MI).Culture plate is hatched 1.5-2 hour then.With being added with 30mM HEPES, Ham ' the s F12 of pH 7.5 preparation contained 1: 3 or 10 IC of the diluent of 1: 5 agonist compounds ₅₀(20nM removes arginine 10-kallidins final concentration, EC for chemical compound plate and gaonist activator plate ₈₀).After coelenterazine is hatched, with automatic flash light emission meter platform the B1 agonist compounds is distributed in the cell plates, the CCD camera that is positioned under the cell plates is taken 12 photos of cell plates with 5 seconds interval, to determine whether these chemical compounds have any gaonist activity.Then, go arginine 10-kallidins to join in the cell plates hB1 gaonist, write down 12 photos again to determine the IC of these antagonisies ₅₀

B. the in vitro tests of the hB2 function of receptors that carries out with the calcium flux:

Use (Wellesley, MA available from PerkinElmer; Article number: hB2 recombinant cell lines (CHO-K1) RBHB2C000EA), go up the inductive intracellular Ca2+ flux of analysis hB2 receptor activation at photofulorography plate readout instrument (FLIPR).Cell culture wherein contains Ham ' s F 12 nutritional blends (InvitrogenCorp., Carlsbad, CA in the T225 flask; Article number: 11765-047), 10% tire clone II Ox blood serum (HyClone, Logan, UT; Article number: SH3006603), (100mM stores liquid to the 1mM Sodium Pyruvate, Invitrogen Corp., article number: 12454-013) and 0.4mg/ml Geneticin (G418; The active Geneticin of 50mg/ml, Invitrogen, article number: 10131-207).Example of spatial compartmentalizationis.FLIPR tested preceding 24 hours, with PBS (Invitrogen) washing hB2/CHO cell once, adding 10ml Versene in every flask (1: 5000, Invitrogen, article number: 15040-066).37 ℃ hatch 5 minutes after, remove Versene, cell is separated from flask, be resuspended in the culture medium.Counting cells is inoculated into 96 hole bread board (Costar, Acton, the MA that the bottom transparent side surfaces is black with 25,000 cells/well; Article number: 3904).Cell is at 37 ℃ of CO ₂Overnight incubation in the incubator.

Culture medium is siphoned away from cell, change with 65 μ l dyeing buffer.This dyeing buffer is the 0.5mM Fluo-4AM storage liquid (MolecularProbes that will be dissolved among the DMSO (contain 10%[w/v] pluronic acid), Eugene, OR) being diluted to concentration in the Eagle culture medium (DMEM) of Clear Dulbecco ' s improvement is that 1 μ M prepares, described DMEM contains 0.1%BSA, 20mM HEPES and 2.5mM probenecid (probenecid suppresses the proteic activity of anion transport, therefore increases the dyeing of cell).Cell was room temperature dyeing 1 hour.With test buffer washed cell twice, so that unnecessary dye liquor is removed.Described test buffer is made up of Hank ' the s balanced salt solution (HBSS) that contains 20mM HEPES, 0.1%BSA and 2.5mM probenecid.After the washing step, remaining volume is 100 μ L in each hole, and cell plates can detect in the FLIPR system.Contained 1: 3 or single-point (10u M final concentration) POC agonist compounds plate or 10 IC of 1: 5 antagonist diluent with the test buffer preparation ₅₀Chemical compound plate, and gaonist activator plate (0.3nM Kallidin I final concentration, EC ₈₀).Cell plates and chemical compound plate are added among the FLIPR, in process of the test, in all 96 holes of cell plates, carry out the fluorescence reading simultaneously.Read 10 readings 1 second the time to set up the steady baseline in each hole, from B1 antagonist plate, take out then 25 μ L rapidly (50 μ L/ second) add each hole.To detect fluorescence signal (1 minute) in 1 second the interval, with 6 seconds interval detection (2 minutes), detect 3 minutes altogether to measure these chemical compounds and whether have any gaonist activity then.Add the B2 gaonist then in cell plates, Kallidin I writes down 3 minutes inhibition percentage ratio or IC when determining that antagonist is 10 μ M (POC plate) again ₅₀

With respect to the peptide of the big PEG polymer of coupling, the carrier that in the test of hB1 aequorin, detects-or the IC of PEG coupling peptide ₅₀Being worth average external activity decreases a little.For example, the acetylated form of this peptide shown in peptide shown in the SEQ ID NO:36 and the SEQ ID NO:37 is to the IC of hB1 receptor ₅₀Value be respectively 3.0nM (+/-5nM, n=8) and 3.2nM (+/-3.2nM, n=9).And the identical peptide that is coupled to PEG as described herein proves its IC ₅₀Value increases by 10 times approximately.In hB2FLIPR test, during up to 10 μ M, the peptide inactivation of natural, acetylizad and PEG coupling form.These chemical compounds do not demonstrate the gaonist activity to hB1 or hB2 receptor.

The C.hB1 receptor-binding peptides is based on the in vitro tests of tissue

Test with following vitro human umbilical vein (HUV) contractility and to measure peptide of the present invention and/or carrier-coupling peptide antagonist activities and selectivity bradykinin b 1 receptor.

The blood vessel (endothelium-denuded vessels) that endothelium is exposed is suspended in the 20ml organ bath (organbath), and described organ bath contains the (95%O of oxygenation ₂And 5%CO ₂) and (37 ℃) standard physiological solt solution of preheating, this physiological solt solution has following composition (representing with mM): NaCl 118.0, KCl 4.7, MgSO ₄1.2, CaCl ₂2.5, KH ₂PO ₄1.2, NaHCO ₃25.0 and glucose 11.0 (pH 7.4).With mole displacement NaCl such as KCl, preparation high concentration K+ solution (80mM KCl).Also there is Hoe140 (1 μ M) in the whole test, mergetpa (1 μ M) and captopril (10 μ M), purpose is respectively to prevent the B2 receptor and prevent the peptide degraded.This tissue links to each other with force cell, with record isometric tension (isometric tension), makes its sufficiently long time of balance under the resting tension of optimizing then.Carry out these tests with the semi-automatic organ piece-rate system that has 8 organ bath (each having multi-channel data obtains).At first will organize contact high concentration K+ solution (80mM KCl) to obtain to have the contraction of control.After washing and the 60 minutes balances subsequently, with tissue contact accumulative total progressive concentration with reference to gaonist Lys-desArg9-BK, with obtain do not have (control formulation) have the test compounds of various concentration or with reference to antagonist Lys-desArg9[Leu8]-concentration-response curve during BK (test formulation), test compounds or with reference to antagonist Lys-desArg9[Leu8]-BK added before contact Lys-desArg9-BK in 15 minutes.In each preparation, produced concentration-response curve to Lys-desArg9-BK.

Detected parameter is inductive tensile maximum variation of each gaonist concentration, and the result is expressed as the percent to the contrast response of KCl.Calculate the EC of gaonist with the linear regression analysis of concentration-response curve ₅₀Value (producing the concentration of a half of peak response).Test compounds and Lys-desArg9[Leu8]-antagonist of BK render a service be calculated as the pA2 value (make the concentration-response curve of gaonist be offset to the right 2 times-log concentration), this value is according to Van Rossum (Van Rossum, J.M., integral dose response curve .II. is used for setting up at isolating organ evaluation (the Technique for the making ofdose-response curves in isolated organs and the evaluation of drug parameters) .Arch.Int.Pharmacodyn.Ther. of the technology and the drug parameters of dose response curve, 143:299-330 (1963)) calculate.Only with causing gaonist concentration-response curve to have the antagonist concentration that obviously is offset to calculate the pA2 value to the right.The pA2 value is three meansigma methods ± s.e.m. that measure.Test the significance,statistical of measuring difference with Si Shi t, p＜0.05 thinks to have significance,statistical.

D. the external B1 of peptide and/or coupling peptide suppresses active

Also can in the neuron culture of dorsal root neuroganglion (DRG), prevent the release and the calcium signal transduction of the activated CGRP of B1, P material by detecting each peptide and/or coupling peptide, estimate described peptide and/or coupling peptide effectiveness as B1 activity inhibitor (that is B1 " neutralization ").

The ganglionic neuron culture of dorsal root.From all spinal cord sections of 19 days (E19) big embryo rat, separate the dorsal root neuroganglion one by one under the aseptic condition, described embryo rat is (the Charles River that the operation separation obtains from the uterus of synchronous pregnancy, the lethal Sprague-Dawley rat of anesthesia, Wilmington, MA).(NY) the middle DRG that collects removes any loose connective tissue and blood vessel for GibcoBRL, Grand Island in the ice-cold L-15 culture medium that contains 5% heat-inactivated horse serum (GibcoBRL).Do not containing Ca ²⁺And Mg ²⁺Dulbecco ' s phosphate buffered saline (PBS) (DPBS), washing DRG is twice among the pH7.4 (GibcoBRL).Dissociating with papain then, (Worthington Biochemical Corp., Freehold NJ) dissociates to DRG in the single-cell suspension liquid in system.In brief, in digestion solution (containing the 20U/ml papain that is dissolved in Earle ' s balanced salt solution (EBSS)), hatched DRG50 minute under 37 ℃.In the culture medium of being made up of MEM/Ham ' sF12 (1: 1), 1mg/ml ovoinhibitor, 1mg/ml ovalbumin and 0.005% deoxyribonuclease I (DNase) of dissociating, the Pasteur pipet grinding of handling through fire with the surface makes cell dissociation.Dissociated cell centrifugal 5 minutes at 200xg is resuspended among the EBSS (containing 1mg/ml ovoinhibitor, 1mg/ml ovalbumin and 0.005%DNase).200xg centrifuge cell suspension is 6 minutes in the gradient solution that contains 10mg/ml ovoinhibitor, 10mg/ml ovalbumin, to remove cell debris, (Fisher Scientific, Pittsburgh PA) filter to remove any agglomerate nylon mesh by 88-μ m then.Measure cell number with hematimeter, with 10 * 10 ³(Sigma, St.Louis MO) and in the 96-orifice plate of 1 μ g/ml mice laminin (GibcoBRL)-Bao quilt, cultivate in complete medium for cells/well seeds cells into 100 μ g/ml poly--ornithine.Complete medium is made up of minimum basal medium (MEM) and Ham ' s F12 (1: 1), penicillin (100U/ml), streptomycin (100 μ g/ml) and 10% heat-inactivated horse serum (GibcoBRL).This culture remains on 37 ℃, 5%CO ₂With 100% humidity.In order to control the growth of non-neuronal cell, contain 5-fluoro-2 '-BrdU (75 μ M) and uridin (180 μ M) in the culture medium.

With B1 and anti-B1 peptide and/or anti--B1 coupling peptide processing.Behind the bed board 2 hours, handle cell with the recombined human β-B1 or the recombinant rat β-B1 of 10ng/ml (0.38nM) concentration.In each culture plate, add the anti--B1 antibody (R﹠amp that contains serial dilution; D Systems, Minneapolis, positive control MN).The coupling peptide (for example, from embodiment 1 peptide or coupling peptide) that adds test peptides or test with 10 kinds of concentration of 3.16 times of serial dilutions.Before adding in the culture, all samples all are diluted in the complete medium.Incubation time is about 40 hours usually, detects VR1 then and expresses.

The mensuration that VR1 expresses in the DRG neuron.With 4% the paraformaldehyde that is dissolved in Hanks ' balanced salt solution culture is fixed 15 minutes, with Superblock (Pierce, Rockford, IL) prevent, changed processing thoroughly 1 hour with the 0.25%Nonidet P-40 (Sigma) that is dissolved in Tris.HCl (Sigma)-buffered saline (TBS) under the room temperature then.With the TBS washed cell that contains 0.1%Tween 20 (Sigma) once, room temperature and rabbit be anti--and VRl IgG (by the Amgen preparation) hatched 1-1.5 hour, (Wallac Oy, Turku Finland) are hatched 1 hour to the anti-rabbit second antibody of room temperature and Eu-labelling then.After each and the antibody incubation, wash (3 * 5 minutes, slowly shake) with TBS.In culture, add and strengthen solution (150 μ l/ holes, Wallac Oy).In time-resolved fluorescence meter (Wallac Oy), detect fluorescence signal then.Compare the expression of VR1 in the sample of determining to handle with described carrier-coupling peptide by standard curve with 0-1000ng/ml B1 titre.By comparing, determine the percent inhibition (suppressing to compare) that B1 expresses VR1 in the DRG neuron with maximum possible with the contrast of handling without B1.

The PEG group of the receptors bind of the link coupled B1 peptide antagonists of each PEG and the weakening of functional activity and interpolation big or small directly related caused about 5-200 reduction doubly to effectiveness.The polyglycine joint of about 5-7 residue can be preserved (the B1 peptide antagonists) function well, and longer joint demonstrates improvement (" flexible joint ") hardly or prove that it has infringement (" rigid joint ") to activity.At last, table 8 has illustrated the width that the application contains with the link coupled B1 peptide antagonists of multiple different PEG.

Table 8: the peptide antagonists of peptide and PEGization is renderd a service (IC to the binding affinity (Ki) and the function of people B1 receptor (hB1) ₅₀)

Activatory PEG reagent	Peptide/PEG	Peptide (X 1)-(Y 1) 0 or1	hB1 Ki (nM)	hB1 IC 50 (nM)
					The MeO-20K-maleimide a	1	SEQ ID NO：28	114	110
The MeO-20K-maleimide a	1	SEQ ID NO：29	252	237
					The MeO-20K-maleimide a	1	SEQ ID NO：30	230	61
The MeO-20K-maleimide a	1	SEQ ID NOS：29+42	22	54
					The MeO-20K-maleimide a	1	SEQ ID NO：32	9	69
The MeO-20K-maleimide a	1	SEQ ID NO：33	75	98
					The MeO-20K-propionic aldehyde b	1	SEQ ID NO：13	1	35
Maleimide-20K-maleimide a	2	SEQ ID NO：33	11	77
					MeO-20K SPA c	1	SEQ ID NO：13	10	52
Tetrakis-20K-SPA c	4	SEQ ID NO：13	0.14	10
					Tetrakis-20K-SPA d	4	SEQ ID NO：13	0.14	10
Do not have	NA	SEQ ID NO：13	0.10	0.5
					Do not have	NA	SEQ ID NO：15	0.19	1
Do not have	NA	SEQ ID NO：49	0.77	0.5
					Do not have	NA	SEQ ID NO：50	0.2	6
Do not have	NA	SEQ ID NO：22	0.38	2
					Do not have	NA	SEQ ID NO：37	0.8	1

A: produce with the method described in the scheme 4 in single jar of reaction (one-pot);

B: carry out reductive amination at the N-terminal residue, ε amine;

C: at N-terminal residue acidylate, ε amine;

D: at N-terminal residue acidylate, α amine

Embodiment 7: the mensuration of peptide and/or coupling stabilized peptide

A. rat kidney brush border microvillus test

Prepare kidney barrier film (kidney membrane) according to (Biochemical Journal, 142:575 (1974)) described methods such as Booth.Measure protein concentration with Bradford (Anal.Biochemistry., 72:248-254 (1976)) method.

B. rat or people's lung S9 homogenate test

Lung as preparation rat or people as described in (Biochemical Pharmacology 33:3471 (1984)) such as Skidgel.

The test compounds of preparation 1mM concentration in PBS solution (pH=7.1).Test compounds is added to organize (proteic final concentration is 2mg/ml) in the goods that obtains among method A or the B, hatches for 37 ℃.At a plurality of time points, with acetonitrile, be dissolved in the 0.1M HCl of acetonitrile or water-soluble 10%TFA protein precipitation.Centrifugal albumen is removed, filter liquor is the membrane filtration by 0.1 μ M again.Use reversed-phase HPLC (4.6 * 300mmNovapak HR C, 18 (Waters Corporation then, Milford, MA), flow velocity=1mL/min, linear gradient from 10%ACN (0.1% formic acid)-90% water (0.1% formic acid) to 50%ACN (0.1% formic acid)-50% water (0.1% formic acid, 20 minutes), use Mass Spectrometer Method.With respect to the internal standard product, the concentration of test compounds is fit to the one-level loss function (first order loss function) ([chemical compound] during with time T _t=[chemical compound] 0 (1-e ^(-kt)); The concentration of test compounds when " [chemical compound] 0 " and " [chemical compound] t " is the concentration of test compounds when being zero time and sampling respectively; Variable " t " is the time of sample analysis; K is the speed of test compounds concentration change).Variable " k " is to measure with the non-linear regression method that JMP statistics software kit provides.The concentration of supposing test compounds is along with time decreased, and ' k ' value is minus.Calculate the half-life with following formula from " k " value that gets self model: T =(Ln 2)/k.

Embodiment 8: anti-nociceptive pain activity in anti--B1 peptide and the body of the link coupled anti-B1-peptide of carrier in rat and monkey pain model

A. remove Mus neuropathic pain model.As Kim and Chung (the experimental model .Pain 50:355-363 that is connected the nervus peripheralis sexually transmitted disease (STD) change that causes in the rat by spinal cord section nerve, (1992)) at first describe, with isoflurane inhalation anesthesia method male Sprague-Dawley rat (200g) is anaesthetized, the left side spinal column Spinal nerve of L5 and L6 level is close to (stitching of 4-0 silk) in the tip of dorsal root ganglion, before sciatic nerve.Close incisions is restored rat.This method causes mechanicalness (palpable) allodynia at left back pawl, this is that affected claw when being recorded in much pressure (with the position homonymy of nerve injury) is estimated from stimulation (the von Frey filaments 4.0-148.1mN) withdrawal of each grade, and the stimulation of described each grade is observed the sole of the foot face (between the palmula) that vertically is applied to claw that covers by metal mesh opening.As Chaplan, S.R., wait (the quantitative assessment .J.Neurosci.Meth of palpable allodynia in the rat claw, 53:55-63 (1994)) described, increase continuously and reduce stimulus intensity and test analytical data, determine the pawl thresholding (PWT) of withdrawing with the Dixon nonparametric.

Normal rat and sham-operation rat (separated neural but is not connected) stand at least 148.1mN (being equivalent to 15g) pressure and less than reacting.The pressure that the rat that spinal nerves connects responds on its affected pawl is little of 4.0mN (being equivalent to 0.41g).Having only those not show the rat that dyskinesias (for example claw dilatory and sagging) and PWT be lower than 39.2mN (being equivalent to 4.0g) just is included in this research.After the operation at least 7 days, handle rat once by s.c. injection test peptides or test carrier coupling peptide (screen dosage usually and be about 1mg/kg and 60mg/kg respectively) or contrast diluent (PBS), after this measure PWT every day, measure 7 days.

B. rat CFA inflammatory pain model.With the slight anesthesia of male Sprague-Dawley rat (200g), 0.15ml injects left back pawl with complete Freund's adjuvant (CFA) with isoflurane inhalation anesthesia method.This method causes mechanicalness (palpable) allodynia at left back pawl, this is that the claw (with the position homonymy of nerve injury) of irriate when being recorded in much pressure is estimated from stimulation (the von Frey filaments 4.0-148.1mN) withdrawal of each grade, and the stimulation of described each grade is observed the sole of the foot face (between the palmula) that vertically is applied to claw that covers by metal mesh opening.As described in (1994) such as Chaplan, increase continuously and reduce stimulus intensity and test and analyze the withdrawal data with the Dixon nonparametric, determine PWT.Having only those not show the rat that dyskinesias (for example claw dilatory and sagging) or skin injury and PWT be lower than 39.2mN (being equivalent to 4.0g) just is included in this research.After the CFA injection at least 7 days, handle rat once by s.c. injection test peptides or test carrier coupling peptide (screening dosage usually is 60mg/kg) or contrast solution (PBS), after this measure PWT every day, measure 7 days.Use following formula: %MPE=100* (PWT of the PWT-control rats of treated rat)/(PWT of 15-control rats), average pawl withdrawal thresholding (PWT) is converted to the percent (%MPE) of maximum possible effect.Therefore, the cutoff value of 15g (148.1mN) is equivalent to 100% MPE, and control reaction is equivalent to 0% MPE.

The preferred peptide of expection the present invention produces with 60mg/kg at about 1mg/kg respectively with carrier-coupling peptide has the related anti-nociceptive pain effect of PD.

B. grivet LPS inflammatory model.Can be substantially as described in deBlois and the Horlick (British Journal ofPharmacology.132:327-335 (2002)) (including this paper in as a reference in full), use by the male grivet of B1 gaonist local assault (Cercopithaecus aethiops St Kitts) and estimate peptide and/or coupling peptide effectiveness as the B1 activity inhibitor.

Whether suppress the inductive edema of B1 in order to measure PEG-coupling peptide antagonists of the present invention, the male grivet of (St Kitts, West Indies) (Cercopithaecus aethiops StKitts) has carried out following research on one's body in CaribbeanPrimates Ltd. experimental farm.Experimental technique be through CR-CHUM (Montreal, Canada) and the animal of Caribbean Primates Ltd. (St Kitts, West Indies) be concerned about that committee checks and accept.Animal Anesthesia (50mg ketamine kg with body weight 6.0 ± 0.5kg (n=67) ^-1), come the pretreatment animal with single intravenous injection LPS (90 μ gkg-1) or by saphena pump pickle (1ml).

1. inflammation research

Test with the veutro skin fold and to estimate the inductive edema of kassinin kinin (Sciberras etc., 1987).In brief, with animal (the 1mg kg of captopril injection through anesthesia ^-1, tested preceding 30 minutes).At veutro zone single subcutaneous injection dKD, BK or carrier (the 2mM amastatin is dissolved in the 100 μ l Ringer ' s lactates).With increase 30-45 minute of calibrated caliper detection of skin pleat thickness.The result be expressed as before the subcutaneous injection and subcutaneous injection after skin fold thickness poor.Captopril and amastatin are respectively applied for and reduce kassinin kinin carboxyl and aminoterminal degraded.

Antagonist SCHILD analyzes

When not having or existing the PEG-peptide antagonists of variable concentrations, after giving LPS, measure the dose response relation of the inductive edema of dKD (1-100nmol) 24 hours the time.BK (30nmol) is as positive control.

The antagonist time course

Single bullet administration (bolus administration) back was measured the time course of the inhibition of antagonist in 4,24,48,72 and/or 96 hours.BK (30nmol) is as positive control.

Medicine

Ketamine hydrochloride, LPS, amastatin and captopril available from Sigma (MO, U.S.A.).All peptides all derive from Phoenix Pharmaceuticals (CA, U.S.A.).

Statistical data

Numeric representation is meansigma methods ± meansigma methods standard error (s.e. of meansigma methods).In edema research, deduct the thickness of the preceding skin fold of injection the skin fold thickness after subcutaneous attack.Carry out curve fitting and calculate EC with Delta Graph 4.0 softwares that are suitable for Apple Computers ₅₀Two-way analysis with variance comes comparing data, carries out azygous single tail Si Shi t test then, adopts Bonferroni to proofread and correct.P＜0.05 thinks to have significance,statistical.

Form in the test in edema, give grivet with LPS and make them begin to increase from zero level B1 receptor gaonist sensitivity.The ground of comparing, uninfluenced to the response of B2 receptor gaonist BK.

It is shocking, the representative 5kD PEG coupling peptide and the 20kD PEG coupling peptide of the peptide analogues that single subcutaneous injection 10mg/kg is identical just are enough to alleviate the inductive inflammatory reaction of having set up of B1 gaonist, and gaonist attack every day that suppresses subsequently was respectively 3 days and 4 days.In 96 hours, do not observe the tachyphylaxis (tachyphalaxis) that B1 is attacked.This effect also is measured as B1 but not B2 has selectivity.And 5K PEG-conjugate is attacked in response to dKD and time of suppressing edema is longer than not link coupled (natural) peptide, but two kinds of molecules all play a role rapidly and be all suitable 1.25 hours effectiveness.

Embodiment 9: the rat pharmacokinetic

By intravenous (iv) or subcutaneous (sc) approach, give male Sprague-Dawley rat with bolus with various peptides or coupling peptide (in aqueous medium).Collect blood sample at a plurality of time points (for example, the injection back is 0,15,30 minute and/or 1,2,4,6,8,10,12,18,24,30,36,42,48,60,72,84,96,120,240 and/or 320 hour) and place heparinization (heparized) pipe.The centrifugal blood plasma of from cell mass, removing, freezing or processing immediately.With compound of interest in analyte-specificity LC-MS/MS or the ELISA method quantitative assay blood plasma.Calculate various standard pharmacokinetic parameters with non-districtization (non-compartmental) method, as clearance rate (CL), apparent clearance rate (CL/F), volume of distribution (Vss), mean residence time (MRT), area under curve (AUC) and t1/2 (t1/2) (for example seeing Table 9).

Table 9: the pharmacokinetic of the peptide B1 antagonist of peptide and PEGization in the rat

Activatory PEG reagent	Peptide/PEG	Peptide	t 1/2 (h)	AUC 0-inf
					The MeO-20K-maleimide a	1	SEQ ID NO：33	-	28387
The MeO-20K-propionic aldehyde b	1	SEQ ID NO：13	33.7 c	16877 c
					Maleimide-20K-maleimide a	2	SEQ ID NO：33	25.6 c	28580 c
MeO-20K SPA d	1	SEQ ID NO：13	27.3 c	10701 c
					Tetrakis-20K-SPA d	4	SEQ ID NO：13	28.7 e	8475 e
Tetrakis-20K-SPA f	4	SEQ ID NO：13	30.8 e	63239 e
					Do not have	NA	SEQ ID NO：13	1.2g	255g
Do not have	NA	SEQ ID NO：15	2.76 c	7720 c
					Do not have	NA	SEQ ID NO：13	0.6 h	9 h
Do not have	NA	SEQ ID NO：49	1.0 i	13862 i
					Do not have	NA	SEQ ID NO：50	2.0 i	5529 i
Do not have	NA	SEQ ID NO：22	1.0 i	7891 i
					Do not have	NA	SEQ ID NO：37	0.4 i	1122 i
Do not have	NA	SEQ ID NO：37	0.6 g	18288 g

A: produced by scheme 4 described methods in single jar of reaction (one-pot).

B:N terminal residue reductive amination, ε amine;

c：1mpk sc；

D:N terminal residue acidylate, ε amine;

e：0.5mpk sc；

F:N terminal residue acidylate, α amine;

g：30mpk sc；h：1mpk iv；i：3mpk iv

Should be understood that and to carry out multiple variation aforesaid the present invention.Therefore, scope of the present invention is limited by following claim.

Sequence table

＜110〉Amgen Inc. (Amgen Inc.)

＜120〉bradykinin b 1 receptor antagonist

<130>A-836B

＜140〉record not

<141>2004-10-22

<150>60/538,929

<151>2004-01-24

＜150〉do not give

<151>2004-10-21

<150>60/513,913

<151>2003-10-22

<160>60

<170>PatentIn version 3.2

<210>1

<211>9

<212>PRT

＜213〉people

<400>1

Arg Pro Pro Gly Phe Ser Pro Phe Arg

1 5

<210>2

<211>10

<212>PRT

＜213〉people

<400>2

Lys Arg Pro Pro Gly Phe Ser Pro Phe Arg

1 5 10

<210>3

<211>11

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<400>3

Met Lys Arg Pro Pro Gly Phe Ser Pro Phe Arg

1 5 10

<210>4

<211>8

<212>PRT

＜213〉people

<400>4

Arg Pro Pro Gly Phe Ser Pro Phe

1 5

<210>5

<211>8

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<400>5

Arg Pro Pro Gly Phe Ser Pro Leu

1 5

<210>6

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<400>6

Lys Arg Pro Pro Gly Phe Ser Pro Leu

1 5

<210>7

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa at 1 place, position is defined as DArg

<220>

<221>misc_feature

<222>(4)..(4)

＜223〉Xaa at 4 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(6)..(6)

＜223〉Xaa at 6 places, position is defined as Thi

<220>

<221>misc_feature

<222>(8)..(8)

＜223〉Xaa at 8 places, position is defined as DTic

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as Oic

<400>7

Xaa Arg Pro Xaa Gly Xaa Ser Xaa Xaa Arg

1 5 10

<210>8

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa at 1 place, position is defined as DArg

<220>

<221>misc_feature

<222>(4)..(4)

＜223〉Xaa at 4 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(6)..(6)

＜223〉Xaa at 6 places, position is defined as Thi

<220>

<221>misc_feature

<222>(8)..(8)

＜223〉Xaa at 8 places, position is defined as DTic

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as Oic

<400>8

Xaa Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5

<210>9

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa at 1 place, position is defined as DArg

<220>

<221>misc_feature

<222>(4)..(4)

＜223〉Xaa at 4 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(6)..(6)

＜223〉Xaa at 6 places, position is defined as Thi

<220>

<221>misc_feature

<222>(8)..(8)

＜223〉Xaa at 8 places, position is defined as DHpe

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as Oic

<400>9

Xaa Arg Pro Xaa Gly Xaa Ser Xaa Xaa Arg

1 5 10

<210>10

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>MOD_RES

<222>(1)..(1)

＜223〉acetylation

<220>

<221>misc_feature

<222>(7)..(7)

＜223〉Xaa at 7 places, position is defined as MePhe

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as D-Beta-NaI

<400>10

Leu Leu Arg Pro Pro Gly Xaa Ser Xaa Ile

1 5 10

<210>11

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa at 1 place, position is defined as DArg

<220>

<221>misc_feature

<222>(4)..(4)

＜223〉Xaa at 4 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(6)..(6)

＜223〉Xaa at 6 places, position is defined as Igl

<220>

<221>misc_feature

<222>(8)..(8)

＜223〉Xaa at 8 places, position is defined as DIgl

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as Oic

<400>11

Xaa Arg Pro Xaa Gly Xaa Ser Xaa Xaa Arg

1 5 10

<210>12

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa at 5 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(7)..(7)

＜223〉Xaa at 7 places, position is defined as Igl

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as DIgl

<220>

<221>misc_feature

<222>(10)..(10)

＜223〉Xaa at 10 places, position is defined as Oic

<400>12

Lys Lys Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>13

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa at 5 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(7)..(7)

＜223〉Xaa at 7 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as Dtic

<220>

<221>misc_feature

<222>(10)..(10)

＜223〉Xaa at 10 places, position is defined as Cpg

<400>13

Lys Lys Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>14

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa at 1 place, position is defined as DArg

<220>

<221>misc_feature

<222>(4)..(4)

＜223〉Xaa at 4 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(6)..(6)

＜223〉Xaa at 6 places, position is defined as Igl

<220>

<221>misc_feature

<222>(8)..(8)

＜223〉Xaa at 8 places, position is defined as Df5f

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as Igl

<400>14

Xaa Arg Pro Xaa Gly Xaa Ser Xaa Xaa Arg

1 5 10

<210>15

<211>10

<212>PRT

＜213〉artificial peptide

<220>

＜223〉artificial

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa at 1 place, position is defined as DOrn

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa at 5 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(7)..(7)

＜223〉Xaa at 7 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as Dtic

<220>

<221>misc_feature

<222>(10)..(10)

＜223〉Xaa at 10 places, position is defined as Cpg

<400>15

Xaa Leu Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>16

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa at 1 place, position is defined as DOrn

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa at 5 places, position is defined as Thz

<220>

<221>misc_feature

<222>(7)..(7)

＜223〉Xaa at 7 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as Dtic

<220>

<221>misc_feature

<222>(10)..(10)

＜223〉Xaa at 10 places, position is defined as Cpg

<400>16

Xaa Leu Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>17

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa at 1 place, position is defined as 3Pal

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa at 5 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(7)..(7)

＜223〉Xaa at 7 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as Dtic

<220>

<221>misc_feature

<222>(10)..(10)

＜223〉Xaa at 10 places, position is defined as Cpg

<400>17

Xaa Leu Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>18

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa at 1 place, position is defined as 4Pal

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa at 5 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(7)..(7)

＜223〉Xaa at 7 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as Dtic

<220>

<221>misc_feature

<222>(10)..(10)

＜223〉Xaa at 10 places, position is defined as Cpg

<400>18

Xaa Leu Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>19

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa at 1 place, position is defined as Cha

<220>

<221>misc_feature

<222>(4)..(4)

＜223〉Xaa at 4 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(6)..(6)

＜223〉Xaa at 6 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(8)..(8)

＜223〉Xaa at 8 places, position is defined as DTic

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as Cpg

<400>19

Xaa Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5

<210>20

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa at 1 place, position is defined as 2Nal

<220>

<221>misc_feature

<222>(4)..(4)

＜223〉Xaa at 4 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(6)..(6)

＜223〉Xaa at 6 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(8)..(8)

＜223〉Xaa at 8 places, position is defined as DTic

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as Cpg

<400>20

Xaa Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5

<210>21

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(4)..(4)

＜223〉Xaa at 4 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(6)..(6)

＜223〉Xaa at 6 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(8)..(8)

＜223〉Xaa at 8 places, position is defined as DTic

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as Cpg

<400>21

Leu Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5

<210>22

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa at 1 place, position is defined as DLys

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa at 5 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(7)..(7)

＜223〉Xaa at 7 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as Dtic

<220>

<221>misc_feature

<222>(10)..(10)

＜223〉Xaa at 10 places, position is defined as Cpg

<400>22

Xaa Leu Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>23

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(2)..(2)

＜223〉Xaa at 2 places, position is defined as DOrn

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa at 5 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(7)..(7)

＜223〉Xaa at 7 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as Dtic

<220>

<221>misc_feature

<222>(10)..(10)

＜223〉Xaa at 10 places, position is defined as Cpg

<400>23

Leu Xaa Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>24

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(2)..(2)

＜223〉Xaa at 2 places, position is defined as Cha

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa at 5 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(7)..(7)

＜223〉Xaa at 7 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as Dtic

<220>

<221>misc_feature

<222>(10)..(10)

＜223〉Xaa at 10 places, position is defined as Cpg

<400>24

Leu Xaa Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>25

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(2)..(2)

＜223〉Xaa at 2 places, position is defined as Abu

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa at 5 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(7)..(7)

＜223〉Xaa at 7 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as Dtic

<220>

<221>misc_feature

<222>(10)..(10)

＜223〉Xaa at 10 places, position is defined as Cpg

<400>25

Leu Xaa Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>26

<211>11

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(2)..(2)

＜223〉Xaa at 1 place, position is defined as 2Nal

<220>

<221>misc_feature

<222>(3)..(3)

＜223〉Xaa can be any naturally occurring aminoacid

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa at 5 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(6)..(6)

＜223〉Xaa can be any naturally occurring aminoacid

<220>

<221>misc_feature

<222>(7)..(7)

＜223〉Xaa at 7 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(8)..(8)

＜223〉Xaa can be any naturally occurring aminoacid

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as Dtic

<220>

<221>misc_feature

<222>(10)..(10)

＜223〉Xaa at 10 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(11)..(1)

＜223〉Xaa can be any naturally occurring aminoacid

<400>26

Leu Xaa Xaa Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>27

<211>13

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<400>27

Cys Gly Gly Gly Lys Arg Pro Pro Gly Phe Ser Pro Leu

1 5 10

<210>28

<211>15

<212>PRT

＜213〉artificial peptide

<220>

＜223〉artificial

<400>28

Cys Gly Gly Gly Gly Gly Lys Arg Pro Pro Gly Phe Ser Pro Leu

1 5 10 15

<210>29

<211>15

<212>PRT

＜213〉artificial peptide

<220>

＜223〉artificial

<400>29

Cys Gly Gly Gly Gly Gly Lys Lys Arg Pro Gly Phe Ser Pro Leu

1 5 10 15

<210>30

<211>17

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<400>30

Cys Gly Gly Gly Gly Gly Lys Arg Lys Arg Pro Pro Gly Phe Ser Pro

1 5 10 15

Leu

<210>31

<211>12

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(3)..(3)

＜223〉Xaa at 3 places, position is defined as CH2-CH2-CH2-CH2-CH2-CH2

<400>31

Cys Gly Xaa Lys Arg Pro Pro Gly Phe Ser Pro Leu

1 5 10

<210>32

<211>16

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(13)..(13)

＜223〉Xaa at 13 places, position is defined as MePhe

<220>

<221>misc_feature

<222>(15)..(15)

＜223〉Xaa at 15 places, position is defined as D-Beta-NaI

<400>32

Cys Gly Gly Gly Gly Gly Leu Leu Arg Pro Pro Gly Xaa Ser Xaa Ile

1 5 10 15

<210>33

<211>16

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(11)..(11)

＜223〉Xaa at 11 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(13)..(13)

＜223〉Xaa at 13 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(15)..(15)

＜223〉Xaa at 15 places, position is defined as Dtic

<220>

<221>misc_feature

<222>(16)..(16)

＜223〉Xaa at 16 places, position is defined as Cpg

<400>33

Cys Gly Gly Gly Gly Gly Lys Lys Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10 15

<210>34

<211>18

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(12)..(12)

＜223〉Xaa at 12 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(13)..(13)

＜223〉Xaa can be any naturally occurring aminoacid

<220>

<221>misc_feature

<222>(14)..(14)

＜223〉Xaa at 14 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(15)..(15)

＜223〉Xaa can be any naturally occurring aminoacid

<220>

<221>misc_feature

<222>(16)..(16)

＜223〉Xaa at 16 places, position is defined as Dtic

<220>

<221>misc_feature

<222>(17)..(17)

＜223〉Xaa at 17 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(18)..(18)

＜223〉Xaa can be any naturally occurring aminoacid

<400>34

Cys Gly Gly Gly Gly Gly Gly Gly Lys Lys Arg Pro Xaa Gly Xaa Ser

1 5 10 15

Xaa Xaa

<210>35

<211>16

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>MOD_RES

<222>(1)..(1)

＜223〉acetylation

<220>

<221>misc_feature

<222>(11)..(11)

＜223〉Xaa can be any naturally occurring aminoacid

<220>

<221>misc_feature

<222>(12)..(12)

＜223〉Xaa at 12 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(13)..(13)

＜223〉Xaa can be any naturally occurring aminoacid

<220>

<221>misc_feature

<222>(14)..(14)

＜223〉Xaa at 14 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(15)..(15)

＜223〉Xaa can be any naturally occurring aminoacid

<220>

<221>misc_feature

<222>(16)..(16)

＜223〉Xaa at 16 places, position is defined as Dtic

<220>

<221>misc_feature

<222>(17)..(17)

＜223〉Xaa at 17 places, position is defined as Cpg

<400>35

Cys Gly Gly Gly Gly Gly Lys Lys Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10 15

<210>36

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa at 5 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(7)..(7)

＜223〉Xaa at 7 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as Dtic

<220>

<221>misc_feature

<222>(10)..(10)

＜223〉Xaa at 10 places, position is defined as Cpg

<400>36

Lys Lys Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>37

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>MOD_RES

<222>(1)..(1)

＜223〉acetylation

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa can be any naturally occurring aminoacid

<220>

<221>misc_feature

<222>(6)..(6)

＜223〉Xaa at 6 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(7)..(7)

＜223〉Xaa can be any naturally occurring aminoacid

<220>

<221>misc_feature

<222>(8)..(8)

＜223〉Xaa at 8 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa can be any naturally occurring aminoacid

<220>

<221>misc_feature

<222>(10)..(10)

＜223〉Xaa at 10 places, position is defined as Dtic

<220>

<221>misc_feature

<222>(11)..(11)

＜223〉Xaa at 11 places, position is defined as Cpg

<400>37

Lys Lys Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>38

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<400>38

Cys Lys Arg Pro Pro Gly Phe Ser Pro Leu

1 5 10

<210>39

<211>16

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(7)..(7)

＜223〉Xaa at 7 places, position is defined as DOrn

<220>

<221>misc_feature

<222>(11)..(11)

＜223〉Xaa at 11 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(13)..(13)

＜223〉Xaa at 13 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(15)..(15)

＜223〉Xaa at 15 places, position is defined as Dtic

<220>

<221>misc_feature

<222>(16)..(16)

＜223〉Xaa at 16 places, position is defined as Cpg

<400>39

Cys Gly Gly Gly Gly Gly Xaa Leu Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10 15

<210>40

<211>16

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(7)..(7)

＜223〉Xaa at 7 places, position is defined as DOrn

<220>

<221>misc_feature

<222>(11)..(11)

＜223〉Xaa at 11 places, position is defined as Thz

<220>

<221>misc_feature

<222>(13)..(13)

＜223〉Xaa at 13 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(15)..(15)

＜223〉Xaa at 15 places, position is defined as Dtic

<220>

<221>misc_feature

<222>(16)..(16)

＜223〉Xaa at 16 places, position is defined as Cpg

<400>40

Cys Gly Gly Gly Gly Gly Xaa Leu Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10 15

<210>41

<211>16

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(8)..(8)

＜223〉Xaa at 8 places, position is defined as DOrn

<220>

<221>misc_feature

<222>(11)..(11)

＜223〉Xaa at 11 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(13)..(13)

＜223〉Xaa at 14 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(15)..(15)

＜223〉Xaa at 15 places, position is defined as Dtic

<220>

<221>misc_feature

<222>(16)..(16)

＜223〉Xaa at 16 places, position is defined as Cpg

<400>41

Cys Gly Gly Gly Gly Gly Leu Xaa Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10 15

<210>42

<211>15

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<400>42

Gly Gly Gly Gly Gly Lys Lys Arg Pro Pro Gly Phe Ser Pro Leu

1 5 10 15

<210>43

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa at 1 place, position is defined as D-Dab

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa at 5 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(7)..(7)

＜223〉Xaa at 7 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as Dtic

<220>

<221>misc_feature

<222>(10)..(10)

＜223〉Xaa at 10 places, position is defined as Cpg

<400>43

Xaa Lys Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>44

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>MOD_RES

<222>(1)..(1)

＜223〉acetylation

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa at 1 place, position is defined as D-Dab

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa at 5 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(7)..(7)

＜223〉Xaa at 7 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as Dtic

<220>

<221>misc_feature

<222>(10)..(10)

＜223〉Xaa at 10 places, position is defined as Cpg

<400>44

Xaa Leu Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>45

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa at 1 place, position is defined as DOrn

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa at 5 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(7)..(7)

＜223〉Xaa at 7 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as Dtic

<220>

<221>misc_feature

<222>(10)..(10)

＜223〉Xaa at 10 places, position is defined as Cpg

<400>45

Xaa Leu Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>46

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>MOD_RES

<222>(1)..(1)

＜223〉acetylation

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa at 1 place, position is defined as DOrn

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa at 5 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(7)..(7)

＜223〉Xaa at 7 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as Dtic

<220>

<221>misc_feature

<222>(10)..(10)

＜223〉Xaa at 10 places, position is defined as Cpg

<400>46

Xaa Leu Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>47

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa at 1 place, position is defined as D-3 ' Pal

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa at 5 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(7)..(7)

＜223〉Xaa at 7 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as Dtic

<220>

<221>misc_feature

<222>(10)..(10)

＜223〉Xaa at 10 places, position is defined as Cpg

<400>47

Xaa Leu Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>48

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>MOD_RES

<222>(1)..(1)

＜223〉acetylation

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa at 1 place, position is defined as D-3 ' Pal

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa at 5 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(7)..(7)

＜223〉Xaa at 7 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as Dtic

<220>

<221>misc_feature

<222>(10)..(10)

＜223〉Xaa at 10 places, position is defined as Cpg

<400>48

Xaa Leu Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>49

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa at 1 place, position is defined as D-Lys

<220>

<221>misc_feature

<222>(2)..(2)

＜223〉Xaa at 2 places, position is defined as D-2-Nal

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa at 5 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(7)..(7)

＜223〉Xaa at 7 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as Dtic

<220>

<221>misc_feature

<222>(10)..(10)

＜223〉Xaa at 10 places, position is defined as Cpg

<400>49

Xaa Xaa Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>50

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(2)..(2)

＜223〉Xaa at 2 places, position is defined as D-2-Nal

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa at 5 places, position is defined as Hyp

<220>

<221>misc_feature

<222>(7)..(7)

＜223〉Xaa at 7 places, position is defined as Cpg

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as Dtic

<220>

<221>misc_feature

<222>(10)..(10)

＜223〉Xaa at 10 places, position is defined as Cpg

<400>50

Leu Xaa Are Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>51

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa at 1 place, position is defined as DOrn

<220>

<221>misc_feature

<222>(3)..(3)

＜223〉Xaa at 3 places, position is defined as Oic

<220>

<221>misc_feature

<222>(6)..(6)

＜223〉Xaa at 6 places, position is defined as Me-Phe

<220>

<221>misc_feature

<222>(8)..(8)

＜223〉Xaa at 8 places, position is defined as D-Beta-NaI

<400>51

Xaa Arg Xaa Pro Gly Xaa Ser Xaa Ile

1 5

<210>52

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>MOD_RES

<222>(1)..(1)

＜223〉acetylation

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa at 1 place, position is defined as DOrn

<220>

<221>misc_feature

<222>(3)..(3)

＜223〉Xaa at 3 places, position is defined as Oic

<220>

<221>misc_feature

<222>(6)..(6)

＜223〉Xaa at 6 places, position is defined as Me-Phe

<220>

<221>misc_feature

<222>(8)..(8)

＜223〉Xaa at 8 places, position is defined as D-Beta-NaI

<400>52

Xaa Arg Xaa Pro Gly Xaa Ser Xaa Ile

1 5

<210>53

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa at 1 place, position is defined as DOrn

<220>

<221>misc_feature

<222>(4)..(4)

＜223〉Xaa at 4 places, position is defined as Oic

<220>

<221>misc_feature

<222>(7)..(7)

＜223〉Xaa at 7 places, position is defined as Me-Phe

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as D-Beta-NaI

<400>53

Xaa Leu Arg Xaa Pro Gly Xaa Ser Xaa Ile

1 5 10

<210>54

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>MOD_RES

<222>(1)..(1)

＜223〉acetylation

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa at 1 place, position is defined as DOrn

<220>

<221>misc_feature

<222>(4)..(4)

＜223〉Xaa at 4 places, position is defined as Oic

<220>

<221>misc_feature

<222>(7)..(7)

＜223〉Xaa at 7 places, position is defined as Me-Phe

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa at 9 places, position is defined as D-Beta-NaI

<400>54

Xaa Leu Arg Xaa Pro Gly Xaa Ser Xaa Ile

1 5 10

<210>55

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(8)..(8)

＜223〉Xaa at 8 places, position is defined as Me-Phe

<400>55

Leu Arg Pro Pro Gly Phe Ser Xaa Ile

1 5

<210>56

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>MOD_RES

<222>(1)..(1)

＜223〉acetylation

<220>

<221>misc_feature

<222>(8)..(8)

＜223〉Xaa at 8 places, position is defined as D-Beta-NaI

<400>56

Leu Arg Pro Pro Gly Phe Ser Xaa Ile

1 5

<210>57

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa at 1 place, position is defined as Orn

<220>

<221>misc_feature

<222>(3)..(3)

＜223〉Xaa at 3 places, position is defined as Oic

<220>

<221>misc_feature

<222>(6)..(6)

＜223〉Xaa at 6 places, position is defined as Me-Phe

<220>

<221>misc_feature

<222>(8)..(8)

＜223〉Xaa at 8 places, position is defined as D-Beta-NaI

<400>57

Xaa Arg Xaa Pro G1y Xaa Ser Xaa I1e

1 5

<210>58

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>MOD_RES

<222>(1)..(1)

＜223〉acetylation

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa at 1 place, position is defined as Orn

<220>

<221>misc_feature

<222>(3)..(3)

＜223〉Xaa at 3 places, position is defined as Oic

<220>

<221>misc_feature

<222>(6)..(6)

＜223〉Xaa at 6 places, position is defined as Me-Phe

<220>

<221>misc_feature

<222>(8)..(8)

＜223〉Xaa at 8 places, position is defined as D-Beta-NaI

<400>58

Xaa Arg Xaa Pro Gly Xaa Ser Xaa Ile

1 5

<210>59

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>misc_feature

<222>(3)..(3)

＜223〉Xaa at 3 places, position is defined as Oic

<220>

<221>misc_feature

<222>(6)..(6)

＜223〉Xaa at 6 places, position is defined as Me-Phe

<220>

<221>misc_feature

<222>(8)..(8)

＜223〉Xaa at 8 places, position is defined as D-Beta-NaI

<400>59

Leu Arg Xaa Pro Gly Xaa Ser Xaa Ile

1 5

<210>60

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉artificial peptide

<220>

<221>MOD_RES

<222>(1)..(1)

＜223〉acetylation

<220>

<221>misc_feature

<222>(3)..(3)

＜223〉Xaa at 3 places, position is defined as Oic

<220>

<221>misc_feature

<222>(6)..(6)

＜223〉Xaa at 6 places, position is defined as Me-Phe

<220>

<221>misc_feature

<222>(8)..(8)

＜223〉Xaa at 8 places, position is defined as D-Beta-NaI

<400>60

Leu Arg Xaa Pro Gly Xaa Ser Xaa Ile

1 5

Claims (49)

1. compositions has following formula:

F-[(X ¹)-(Y ¹) _n], or the last acceptable salt of its physiology, wherein:

X ¹And Y ¹Independent separately respectively is formula-L ¹-P ¹With-L ²-P ²Peptide;

F is covalently bonded in X ¹Or Y ¹Carrier;

L ¹And L ²Do not exist, or independently have the joint of 0-9 amino acid residue separately;

N is 0-3; With

P ¹And P ²Independent separately is the bradykinin b 1 receptor peptide antagonists.

2. compositions has following formula:

F '-R _z, or the last acceptable salt of its physiology, wherein:

F ' is the multivalence carrier;

R is independent under each situation to be-(X ¹)-(Y ¹) _n, wherein R is covalently bonded in F ';

X ¹And Y ¹Independent separately respectively is formula-L ¹-P ¹With-L ²-P ²Peptide;

L ¹And L ²Do not exist or independent separately for having the joint of 0-9 amino acid residue;

N is 0-3;

Z is 2-8; With

P ¹And P ²Independent separately is the bradykinin b 1 receptor peptide antagonists.

3. compositions as claimed in claim 1 or 2 is characterized in that P ¹And P ²Independent of separately being selected from SEQID NO:5-26, the bradykinin b 1 receptor peptide antagonists of 43-60 and derivant thereof.

4. compositions as claimed in claim 1 or 2 is characterized in that X ¹It is peptide with the aminoacid sequence that is selected from SEQ ID NO:27-41 and derivant thereof.

5. compositions as claimed in claim 1 or 2 is characterized in that, n is 1 and P ²It is peptide with sequence shown in the SEQ IDNO:42.

6. compositions as claimed in claim 1 or 2 is characterized in that n is 0.

7. compositions as claimed in claim 1 or 2 is characterized in that P ¹And P ²Independent separately is peptide antagonists, and described peptide antagonists is selected from by the defined peptide of following formula:

NH ₂-a ⁰a ¹a ²a ³a ⁴a ⁵a ⁶a ⁷a ⁸a ⁹a ¹⁰a ¹¹a ¹²a ¹³a ¹⁴-COOH

Wherein:

a ⁰Do not exist, or alkalescence or neutral aromatics, aliphatic, heterocycle or alicyclic aminoacid or alkaline dipeptides;

a ¹, a ², a ³And a ⁴Independent separately is alkalescence or neutral aromatics, aliphatic, heterocycle or alicyclic aminoacid;

a ⁶Be Ser;

a ⁵, a ⁷And a ⁸Independent separately is aromatics, aliphatic, heterocycle or alicyclic aminoacid, and condition is a ⁵, a ⁷And a ⁸At least one be selected from Chg, Cpg, Igla, Iglb, Niga and the Nigb of D-or L-configuration; With

a ⁹, a ¹⁰, a ¹¹, a ¹², a ¹³And a ¹⁴Do not exist, or be natural amino acid independently separately.

8. compositions as claimed in claim 7 is characterized in that:

a ⁰Do not exist, or basic amino acid or alkaline dipeptides;

a ¹Be basic amino acid or alkaline dipeptides;

a ²Be Pro;

a ³Be Hyp;

a ⁴Be Gly;

a ⁵And a ⁸Be indanyl aminoacid;

a ⁶Be Ser;

a ⁷Be D-indanyl aminoacid;

a ⁸Be Cpg; With

a ⁹, a ¹⁰, a ¹¹, a ¹², a ¹³And a ¹⁴Do not exist, or be natural amino acid independently separately.

9. compositions as claimed in claim 7 is characterized in that:

a ⁰Be the aminoacid that is selected from down group: Arg, D-Arg, Orn, D-Orn, Lys, Dlys, or by two dipeptides that aminoacid is formed: Arg, the D-Arg, Orn, D-Orn, Lys, the Dlys that are independently selected from down group;

a ¹Be Arg;

a ²Be Pro;

a ³Be Hyp;

a ⁴Be Gly;

a ⁵Be Cpg;

a ⁶Be Ser;

a ⁷Be DTic; With

a ⁸Be Cpg.

10. compositions as claimed in claim 7 is characterized in that:

a ⁰Be Lys-Lys;

a ¹Be Arg;

a ²Be Pro;

a ³Be Hyp;

a ⁴Be Gly;

a ⁵Be Iglb;

a ⁶Be Ser;

a ⁷Be DIglb; With

a ⁸Be Oic.

11. compositions as claimed in claim 7 is characterized in that:

a ⁰Be DArg;

a ¹Be Arg;

a ²Be Pro;

a ³Be Hyp;

a ⁴Be Gly;

a ⁵Be Igl;

a ⁶Be Ser;

a ⁷Be DIgl; With

a ⁸Be Oic.

12. compositions as claimed in claim 1 or 2 is characterized in that, P ¹And P ²Independent separately is peptide antagonists, and described peptide antagonists is selected from by the defined peptide of following formula:

NH ₂-a ⁰a ¹a ²a ³a ⁴a ⁵a ⁶a ⁷a ⁸a ⁹a ¹⁰a ¹¹a ¹²a ¹³a ¹⁴-COOH

Wherein: a ⁰Do not exist, or basic amino acid or alkaline dipeptides;

a ¹Be Arg;

a ²Be Pro;

a ³Be Pro;

a ⁴Be Gly;

a ⁵Be Me-Phe;

a ⁶Be Ser;

a ⁷Be D-β-NaI; With

a ⁸Be Ile.

13. compositions as claimed in claim 1 or 2 is characterized in that, described L ¹Be the peptidyl joint, it has the aminoacid sequence that is selected from SEQ ID NO:100-SEQ ID NO:105.

14. compositions as claimed in claim 3 is characterized in that, described L ¹Be the peptidyl joint, it has the aminoacid sequence that is selected from SEQ ID NO:100-SEQ ID NO:105.

15. a method is used for the treatment of, prevention or alleviation and B1 be active relevant or by the disease or the disease of its mediation, this method comprises that administration of human or animal target treat the claim 1 or the 2 described compositionss of effective dose.

16. a method is used for the treatment of, prevention or alleviation and B1 be active relevant or by the disease or the disease of its mediation, this method comprises that administration of human or animal target treat the described compositions of claim 3 of effective dose.

17. method as claimed in claim 15, it is characterized in that described disease or disease are selected from: the pain that inflammation, inflammatory pain, acute pain, toothache, backache, low back pain, wound cause, operation property pain, inflammatory bowel, asthma and allergic rhinitis.

18. method as claimed in claim 16, it is characterized in that described disease or disease are selected from: the pain that inflammation, inflammatory pain, acute pain, toothache, backache, low back pain, wound cause, operation property pain, inflammatory bowel, asthma and allergic rhinitis.

19. a pharmaceutical composition, described compositions contain claim 1 or 2 described compositionss and at least a pharmaceutically acceptable diluent, excipient or carrier.

20. a compositions has following formula:

F-[(X ¹)-(Y ¹) _n], or the last acceptable salt of its physiology, wherein:

X ¹And Y ¹Independent separately respectively is formula-L ¹-P ¹With-L ²-P ²Peptide;

F is covalently bonded in X ¹Or Y ¹PEG;

L ¹And L ²Do not exist, or independent of separately having the joint of 0-9 amino acid residue;

N is 0-3; With

P ¹And P ²Independent separately is the bradykinin b 1 receptor peptide antagonists.

21. compositions as claimed in claim 2 is characterized in that, described F ' is multivalence PEG.

22., it is characterized in that P as claim 20 or 21 described compositionss ¹And P ²Independent of separately being selected from the bradykinin b 1 receptor peptide antagonists of SEQ ID NO:5-60 and derivant thereof.

23. compositions as claimed in claim 21 is characterized in that, X ¹It is peptide with the aminoacid sequence that is selected from SEQ ID NO:27-41 and derivant thereof.

24. compositions as claimed in claim 21 is characterized in that, n be 0 and Z be 2-8.

25. compositions as claimed in claim 21 is characterized in that, Z is 4.

26. a compositions has following formula:

F-[(X ¹)-(Y ¹) _n], or the last acceptable salt of its physiology, wherein:

X ¹And Y ¹Independent separately respectively is formula-L ¹-P ¹With-L ²-P ²Peptide;

F is covalently bonded in X ¹Or Y ¹Carrier;

L ¹And L ²Do not exist, or independent of separately having the joint of 0-9 amino acid residue;

N is 0-3; With

P ¹And P ²Independent separately is peptide antagonists, and described peptide antagonists is selected from the defined peptide of following formula:

NH ₂-a ⁰a ¹a ²a ³a ⁴a ⁵a ⁶a ⁷a ⁸a ⁹a ¹⁰a ¹¹a ¹²a ¹³a ¹⁴-COOH

Wherein:

a ⁰Do not exist, or alkalescence or neutral aromatics, aliphatic, heterocycle or alicyclic aminoacid or alkaline dipeptides;

a ¹, a ², a ³And a ⁴Independent separately is alkalescence or neutral aromatics, aliphatic, heterocycle or alicyclic aminoacid;

a ⁶Be Ser;

a ⁵, a ⁷And a ⁸Independent separately is aromatics, aliphatic, heterocycle or alicyclic aminoacid, and condition is a ⁵, a ⁷And a ⁸At least one be selected from Chg, Cpg, Igla, Iglb, Niga and the Nigb of D-or L-configuration; With

a ⁹, a ¹⁰, a ¹¹, a ¹², a ¹³And a ¹⁴Do not exist, or be natural amino acid independently separately.

27. a compositions has following formula:

F '-R _z, or the last acceptable salt of its physiology, wherein:

F ' is the multivalence carrier;

R is independent under each situation to be-(X ¹)-(Y ¹) _n, wherein R is covalently bonded in F ';

X ¹And Y ¹Independent separately respectively is formula-L ¹-P ¹With-L ²-P ²Peptide;

L ¹And L ²Do not exist, or independent of separately having the joint of 0-9 amino acid residue;

N is 0-3;

Z is 2-8; With

P ¹And P ²Independent separately is peptide antagonists, and described peptide antagonists is selected from the defined peptide of following formula:

NH ₂-a ⁰a ¹a ²a ³a ⁴a ⁵a ⁶a ⁷a ⁸a ⁹a ¹⁰a ¹¹a ¹²a ¹³a ¹⁴-COOH

Wherein:

a ⁰Do not exist, or alkalescence or neutral aromatics, aliphatic, heterocycle or alicyclic aminoacid or alkaline dipeptides;

a ¹, a ², a ³And a ⁴Independent separately is alkalescence or neutral aromatics, aliphatic, heterocycle or alicyclic aminoacid;

a ⁶Be Ser;

a ⁵, a ⁷And a ⁸Independent separately is aromatics, aliphatic, heterocycle or alicyclic aminoacid, and condition is a ⁵, a ⁷And a ⁸At least one be selected from Chg, Cpg, Igla, Iglb, Niga and the Nigb of D-or L-configuration; With

a ⁹, a ¹⁰, a ¹¹, a ¹², a ¹³And a ¹⁴Do not exist, or be natural amino acid independently separately.

28. as claim 26 or 27 described compositionss, it is characterized in that,

a ⁰Do not exist, or basic amino acid or alkaline dipeptides;

a ¹It is basic amino acid;

a ²Be Pro;

a ³Be Hyp;

a ⁴Be Gly;

a ⁵And a ⁸Independent separately is indanyl aminoacid;

a ⁶Be Ser;

a ⁷Be D-indanyl aminoacid;

a ⁸Be Cpg; With

a ⁹, a ¹⁰, a ¹¹, a ¹², a ¹³And a ¹⁴Do not exist, or be natural amino acid independently separately.

29., it is characterized in that as claim 26 or 27 described compositionss:

a ⁰Be the aminoacid that is selected from down group: Arg, D-Arg, Orn, D-Orn, Lys, Dlys, or by two dipeptides that aminoacid is formed: Arg, the D-Arg, Orn, D-Orn, Lys, the Dlys that are independently selected from down group;

a ¹Be Arg;

a ²Be Pro;

a ³Be Hyp;

a ⁴Be Gly;

a ⁵Be Cpg;

a ⁶Be Ser;

a ⁷Be DTic; With

a ⁸Be Cpg.

30., it is characterized in that as claim 26 or 27 described compositionss:

a ⁰Be D-Arg, or be selected from the dipeptides of Lys-Lys, DLys-Lys and DOrn-Lys;

a ¹Be Arg;

a ²Be Pro;

a ³Be Hyp;

a ⁴Be Gly;

a ⁵Be Iglb;

a ⁶Be Ser;

a ⁷Be DIglb; With

a ⁸Be Oic.

31., it is characterized in that as claim 26 or 27 described compositionss:

a ⁰Be D-Arg or Lys-Lys dipeptides;

a ¹Be Arg;

a ²Be Pro;

a ³Be Hyp;

a ⁴Be Gly;

a ⁵Be Igl;

a ⁶Be Ser;

a ⁷Be DIgl; With

a ⁸Be Oic.

32. compositions as claimed in claim 1 or 2 is characterized in that:

P ¹And P ²Independent separately is peptide antagonists, and described peptide antagonists is selected from by the defined peptide of following formula:

NH ₂-a ⁰a ¹a ²a ³a ⁴a ⁵a ⁶a ⁷a ⁸a ⁹a ¹⁰a ¹¹a ¹²a ¹³a ¹⁴-COOH

Wherein:

a ⁰Be the aminoacid that is selected from down group: Arg, D-Arg, Orn, D-Orn, Lys, Dlys, or by two dipeptides that aminoacid is formed: Arg, the D-Arg, Orn, D-Orn, Lys, the Dlys that are independently selected from down group;

a ¹Be Arg;

a ²Be Pro;

a ³Be Hyp;

a ⁴Be Gly;

a ⁵Be Me-Phe;

a ⁶Be Ser;

a ⁷Be D-β-NaI; With

a ⁸Be Ile.

33. compositions as claimed in claim 32 is characterized in that, described L ¹Be the peptidyl joint, it has the aminoacid sequence that is selected from SEQ ID NO:100-SEQ ID NO:105.

34. compositions as claimed in claim 33 is characterized in that, the molecular weight of described Polyethylene Glycol is selected from down group:

I.5,000 dalton;

Ii.20,000 dalton;

Iii.24,000 dalton;

Iv.30,000 dalton;

V.40,000 dalton; With

Vi.60,000 dalton.

35. compositions as claimed in claim 34 is characterized in that, described compositions can and have in mammalian body the acceptable half-life on the therapeutics at external antagonism B1 receptor active.

36. a method is used for the treatment of, prevention or alleviation and B1 be active relevant or by the disease or the disease of its mediation, this method comprises that administration of human or animal target treat the described compositions of claim 34 of effective dose.

37. method as claimed in claim 35, it is characterized in that described disease or disease are selected from: the pain that inflammation, inflammatory pain, acute pain, toothache, backache, low back pain, wound cause, operation property pain, inflammatory bowel, asthma and allergic rhinitis.

38. a pharmaceutical composition, described compositions contain the described compositions of claim 34 and at least a pharmaceutically acceptable diluent, excipient or carrier.

39. compositions as claimed in claim 34 is used for the treatment of application in the medicine of disease or disease in preparation, described disease or disease are selected from: the pain that inflammation, inflammatory pain, acute pain, toothache, backache, low back pain, wound cause, operation property pain, inflammatory bowel, asthma and allergic rhinitis.

40. a bradykinin b 1 receptor peptide antagonists, described peptide antagonists comprise the aminoacid sequence that is selected from SEQ ID NO:15-35,39-54 and derivant thereof, or its physiology goes up acceptable salt.

41. a bradykinin b 1 receptor peptide antagonists, described peptide antagonists comprises the aminoacid sequence that is selected from SEQ ID NO:15-35, or its physiology goes up acceptable salt.

42. peptide as claimed in claim 41 is characterized in that, it comprises the aminoacid sequence that is selected from SEQ ID NO:15-26.

43. peptide as claimed in claim 40 is characterized in that, described N-terminal aminoacid is D type-aminoacid.

44. peptide as claimed in claim 43 is characterized in that, described N-terminal aminoacid is selected from D-Dab, Dlys, Darg and DOrn.

45. a pharmaceutical composition, it comprises the bradykinin b 1 receptor peptide antagonists, and described peptide antagonists contains aminoacid sequence and at least a pharmaceutically acceptable diluent, excipient or the carrier that is selected from SEQ ID NO:15-35.

46. method, be used for the treatment of, prevention or alleviation and B1 be active relevant or by the disease or the disease of its mediation, this method comprises the pharmaceutical composition of administration of human or animal target treatment effective dose, described pharmaceutical composition comprises the bradykinin b 1 receptor peptide antagonists, and described peptide antagonists contains aminoacid sequence and at least a pharmaceutically acceptable diluent, excipient or the carrier that is selected from SEQ ID NO:15-35.

47. method as claimed in claim 46, it is characterized in that described disease or disease are selected from: the pain that inflammation, inflammatory pain, acute pain, toothache, backache, low back pain, wound cause, operation property pain, inflammatory bowel, asthma and allergic rhinitis.

48. the described pharmaceutical composition of claim 19 is used for the treatment of application in the medicine of disease or disease in preparation, described disease or disease are selected from: the pain that inflammation, inflammatory pain, acute pain, toothache, backache, low back pain, wound cause, operation property pain, inflammatory bowel, asthma and allergic rhinitis.

49. the described pharmaceutical composition of claim 45 is used for the treatment of application in the medicine of disease or disease in preparation, described disease or disease are selected from: the pain that inflammation, inflammatory pain, acute pain, toothache, backache, low back pain, wound cause, operation property pain, inflammatory bowel, asthma and allergic rhinitis.