CN1849132A - Compounds that modulate neuronal growth and their uses - Google Patents

Abstract

Cyclic peptides and peptidomimetics are provided that bind to and/or modulate activities associated with Trk receptors, including processes associated with the growth and repair of the central nervous system (e.g., neuronal growth and survival, axonal growth, neurite outgrowth and synaptic plasticity). Cyclic peptides and peptidomimetics are also provided that block or reduce the effect of other factors that inhibit growth and/or repair of the central nervous system. Pharmaceutical compositions and other formulations comprising these compounds are provided. In addition, the invention provides methods for using the cyclic peptides and peptidomimetics to modulate Trk mediated activities, including processes such as neuronal growth, survival and recover, axonal growth, neurite outgrowth, and synaptic plasticity. Further, the invention provides methods for promoting central nervous system (CNS) neuron growth by administering a p75 receptor binding agent.

Description

Regulate the chemical compound of neure growth and their purposes

1. the cross reference of related application

According to 35U.S.C.119 (e), requirement is enjoyed and being submitted in 10 days U.S. Provisional Patent Application series number 60/501 of JIUYUE in 2003,864, submit in the series number 60/559,898 on April 5th, 2004 and submit in the priority of the series number 60/603,187 on August 20th, 2004.The content of these provisional application all is incorporated herein by reference.

2. invention field

The present invention relates to regulate the compositions and the method for central nervous system (CNS) growth and reparation, comprise process such as neuronal survival, axon growth and synaptic plasticity.More specifically, the present invention relates to be referred to as the agonist of receptor family of Trk receptor or the chemical compound (comprising cyclic peptide and peptide mimics (peptidomimetic) chemical compound) of antagonist, described Trk expression of receptor is in the surface of neuronal cell and these processes of regulating and control the CNS growth and repairing.Further, the present invention relates to use the p75 bonding agent to promote CNS growth and the method for repairing.

3. background of invention

Because the neuronic regeneration capacity in the middle of central nervous system's environment is poor, therefore the damage to central nervous system (CNS) can have destructive consequence.Neuronic regeneration capacity preferably in the middle of this and the peripheral nervous system forms a sharp contrast.Referring to, for example, Horner ﹠amp; Gage, " nature " (Nature) 2000,407:963-970.Numerous disease for example, only gives some instances, and Alzheimer, parkinson disease, apoplexy, head and spinal cord injury are all relevant with the damage of CNS, and this damage is very serious usually, even makes CNS depletion, longer duration or or even nonvolatil.For these state of an illness, do not have Therapeutic Method at present, even lack palliative treatment.

3.1 neurotrophin

The adjusting that known neuronic growth at present and regeneration are subjected to some to be referred to as the polypeptide growth factor of neurotrophin or " NTs " at least in part, they are in conjunction with also activating the cell surface receptor with inherent tyrosine kinase activity.It is believed that the combination once neurotrophin, then these receptors become autophosphorylation on one or more amino acid residues, combine with molecule in the important born of the same parents of signal transduction then.Summary is referring to Ulrich ﹠amp; Schlessinger, Cell 1990,61:203-212.

First certified neurotrophin known in the art is nerve growth factor (NGF), and its sensation and sympathetic neuron to developmental peripheral nervous system has significant effects.Referring to Levi-Montalcini ﹠amp; Angeletti, " physiology's comment " (Physiol.Rev.) 1968,48:534-569; People such as Thoenen, " comment of Physiology and biochemistry pharmacy) " (Rev.Physiol.Biochem.Pharmacol.) 1987,109:145-178; Thoenen ﹠amp; Barde, " physiology's comment " (Physiol.Rev.) 1980,60:1284-1325; Whittemore ﹠amp; Seiger, " brain research " (Brain Res.) 1987,434:439-464; Angeletti ﹠amp; Bradshaw, " NAS's progress " (Proc.Natl.Acad.Sci.U.S.A.) 1971,68:2417-2420; People such as Angeletti, " biochemistry " (Biochemistry) 1973,12:100-115.The lineal congener (orthologs) of NGF was separated and characterize in many other species already, comprised mice, birds, reptile and Fish (people such as Scott, " nature " (Nature) 1983,302:538-540; People such as Schwartz, " neuro chemistry magazine " (J.Neurochem.) 1989,52:1203-1209; With people such as Hallb   k, " neuron " (Neuron) 1991,6:845-858).

Many other NTs also are known in the art.These comprise Brain Derived Neurotrophic Factor (BDNF), also are referred to as neurotrophin-2 (NT-2).Referring to people such as Leibrock, " nature " (Nature) 1989,341:149-152.Also has other NTs, comprise the factor (people such as Ernfors who is called the neuron factor (NF) at first and generally is referred to as neurotrophin-3 or " NT-3 " now, " NAS's progress " (Proc.Natl.Acad.Sci.U.S.A.) 1990,87:5454-5458; People such as H  hn, " nature " (Nature) 1990,344:339; People such as Maisonpierre, " science " (Science) 1990,247:1446; People such as Rosenthal, " neuron " (Neuron) 1990,4:767; Jones ﹠amp; Reichardt, " NAS's progress " (Proc.Natl.Acad.Sci.U.S.A.) 1990,87:8060-8064; And people such as Kaisho, " FEBS communication " (FEBS Lett.) 1990,266:187).Neurotrophin-4 and-5 (NT-4 and NT-5) also is known.Referring to people such as Hallbook, " neuron " (Neuron) 1991,6:845-858; People such as Berkmeier, " neuron " (Neuron) 1991,7:857-866; People such as Ip, " NAS's progress " (Proc.Natl.Acad.Sci.U.S.A.) 1992,89:3060-3064.The U.S. Patent No. 5,364,769 of authorizing Rosenthal on the 15th also referring to December in 1994.The mammal NT-5 molecule of describing in view of people's (ditto) such as Berkmeier afterwards is regarded as the mammal direct line congener of XenoPus (Xenopus) NT-4 that people such as Hallbrook (ditto) describe, so it also generally is referred to as NT-4/5.Fig. 1 provides the comparison of BDNF, NT4, NT3 and NGF among the NT.

3.2Trk receptor

Neurotrophin mediates its effect by tyrosine kinase receptor family, and this receptor family is expressed in the surface of neuronal cell and is commonly referred to as the Trk-receptor.At least three kinds of different Trk-receptors are known and had described in the prior art already: TrkA, TrkB and TrkC.Summary is referring to the U.S. Patent No. 5,844,092,5,877,016,6,025,166,6,027,927 and 6,153,189 that all belongs to people such as Presta.Although the structure of different Trk-receptors is similar with sequence, alternative splicing has increased the complexity of this family, makes every kind of receptor produce several different isoforms.Fig. 2 A-2C provides the comparison of different Trk-receptor amino acid sequences in the literary composition, has listed the consensus sequence and the border in the different structure territory of every kind of receptor.Also referring to U.S. Patent No. 5,877, Figure 16 A-16C in 016.

Although there are some overlapping, every kind of different Trk-receptor shows specific binding affinity to different neurotrophins.Therefore, TrkA is considered to not only in conjunction with NGF, but also in conjunction with NT-3 and NT-4/5 (but not in conjunction with BDNF).TrkB is considered in conjunction with BDNF, NT-3, NT-4 and NT-5, but not in conjunction with NGF.On the contrary, TrkC only is considered in conjunction with NT-3, but any in conjunction with in other neurotrophin not.

Studies confirm that in a large number the Trk-receptor is the treatment target that brain is repaired.Referring to, for example, people such as Liu, J.Neurosci.1999,19:4370-4387; People such as Menei, Eur.J.Neurosci.1998, people such as 10:607-621 and Kobayashi, J.Neurosci.1997,17:9583-9595.Also used the X-radiocrystallgraphy to study Trk-receptor and their part, to obtain the three dimensional structure of ligand-receptor in conjunction with complex.People such as Wiesmann, Nature 1999,401:184-188; People such as Banfield, Structure (Camb) 2001,9:1191-1199.These researchs and other studies show that the dimerization of having induced acceptor monomer in conjunction with the neurotrophin of Trk-receptor, and the result causes the inherent tyrosine kinase activity of this receptor to increase.The activity of this increase has triggered signal transduction cascade successively, it is believed that described signal transduction cascade is of value to neuron by promoting neuronal survival, axon growth and synaptic plasticity.Snider, Cell 1994,77:627-638; Kaplan ﹠amp; Miller, " modern neurobiology progress " (Curr.Opin.Neurobiol.) 2000,10:381-391.

The chemical compound of therefore, can targeting and activating Trk-receptor (Trk-receptor " agonist " just) will be that this viewpoint useful and that be worth exploring has obtained suitable approval.Referring to, for example, people such as Lindsay, " experimental neurology " (Exp.Neurol.) 1993,124:103-188; Olson, " international neuro chemistry " (Neurochem.Int.) 1994,25:1-3.In addition, some neurotrophin (for instance, BDNF) level strengthen also medical condition with for example epileptics relevant (people such as Binder, " neuroscience trend " (Trends Neurosci.) 2001,24:47-53).Therefore, also will be useful even suppress the chemical compound (Trk-receptor " antagonist " just) of Trk-receptor active.Although exist thisly to press for for a long time, these chemical compounds are unintelligible at most.As macromole, the therapeutic of neurotrophin self effect level send constituted sizable, be likely unsurmountable challenge.In addition, natural neurotrophin can with the p75 acceptor interaction in other receptor such as the neuron, this receptor subsides relevant with Neuron Apoptosis and growth cone.People such as Lee, " modern neurobiology progress " (Curr.Opin.Neurobiol.) 2001,11:281-286.

Yet previous is that the peptide mimics agonist of design Trk-receptor and/or the effort of antagonist are not achieved success yet.For example, reported that the cyclic peptide that derives from neurotrophin NGF ring 1 moderately simulates the existence activity of NGF.Yet, these peptides as if with p75 but not the dependent mode of Trk-receptor play a role.People such as Long, " Neuroscience Research magazine " (J.Neurosci.Res.) 1997,48:1-17.It is said that some NGF encircle 4 cyclic peptide and show the NGF sample existence activity of being sealed by the Trk antagonist.Yet, it is reported it only is the 10-15% that reacts by the maximum that NGF neurotrophin self promotes by the maximum existence reaction of those inducing peptides.Referring to people such as Xie, " journal of biological chemistry " (J.Biol.Chem.) 2000, people such as 275:29868-29874 and Maliartchouk, " journal of biological chemistry " (J.Biol.Chem.) 2000,275:9946-9956.The BDNF that had proved bicyclo-and three cyclodimerization bodily form formulas already encircles 2 peptides and has BDNF sample activity.Yet, again, they inductive maximum existence reaction it is reported it only is 30% of the maximum reaction that promotes by natural neurotrophin.People such as O ＇ Leary, " journal of biological chemistry " (J.Biol.Chem.) 2003,278:25738-25744 (electronic publication, on May 2nd, 2003).

Therefore, the compositions that can regulate (just, increasing or inhibition) neure growth and recovery is pressed for lasting the existence for a long time.Process and method (comprising Therapeutic Method) needs to effective adjusting neure growth and recovery also exist.

3.21.p75 receptor neurotrophin receptor

Known p75 receptor plays a role in relevant Neuron Apoptosis and growth inhibiting signal conduction complex.Barker，Neuron 2004，42：529-533。The p75 receptor is the member of tumor lethal factor (TNR) superfamily, and to be rich in domain (CRDs) with the cysteine of the outer part of its born of the same parents be feature.These CRDs are essential to the combination of neurotrophin, and the p75 receptor serves as low-affinity receptor concerning neurotrophin such as NGF, BDNF, NT-3 and NT-4.Huang and Reichardt, " biochemistry yearbook " (Annu.Rev.Biochem.) 2003,72:609-642.NGF, BDNF, NT-3 and NT-4 each other effective competition in conjunction with the p75 receptor.In suppressing environment, these neurotrophins can be used in competition each other to the combination of p75 receptor, only depend on the reaction of Trk signal conduction with announcement.Barker and Shooter, " neuron " (Neuron) 1994,13:203-215.

3.22.p75 receptor is known NGF TDIKGKE motif

The TDIKGKE motif of first beta hairpin ring of known formation NGF is being brought into play crucial effect in the combination of NGF to the p75 receptor.He and Garcia, " science " (Science) 2004,304:870-875; People such as Ibanez, Cell 1992,69:329-341.In addition, the TDIKGKE motif and the p75 acceptor interaction of constraint (constrained), and expection competition neurotrophin is to the combination of this receptor.People such as Longo, " Neuroscience Research magazine " (J.Neurosci.Res.) 1997,48:1-17.

Reported already that the cyclic peptide that derives from NGF ring 1 and peptide mimics chemical compound moderately simulated NGF and promote the activity of neure growth (to see people's such as Saragovi U.S. Patent No. 6,017,878), as and if these peptides are with the dependent mode of the p75 receptor (people such as Longo that plays a role, " Neuroscience Research magazine " (J.Neurosci.Res.) 1997,48:1-17).It is said that some NGF encircle 4 cyclic peptide and show the NGF sample neure growth facilitation of being sealed by the Trk antagonist.Yet, it is reported it only is the 10-15% that reacts by the maximum that NGF neurotrophin self promotes by the maximum reaction of those inducing peptides.See people such as Xie, " journal of biological chemistry " (J.Biol.Chem.) 2000, people such as 275:29868-29874 and Maliartchouk, " journal of biological chemistry " (J.Biol.Chem.) 2000,275:9946-9956.The BDNF that had proved bicyclo-and three cyclodimerization bodily form formulas already encircles 2 peptides and has BDNF sample activity.Yet, again, they inductive maximum reaction it is reported it only is 30% of the maximum reaction that promotes by natural neurotrophin.People such as O ＇ Leary, " journal of biological chemistry " (J.Biol.Chem.) 2003,278:25738-25744 (electronic publication, on May 2nd, 2003).

3.3. inhibition signal

The ability of the limited reparation of central nervous system damage be considered to be at least in part since stop the existence of the inhibition product of axon regeneration-the comprise inhibitor relevant with impaired myelin (Berry, Bibl.Anat.1982,23:1-11).Really, the myelinic biochemical research of central authorities has been identified that two kinds contain cellular invasion and suppress active protein fractions (Caroni; Schwab, " cytobiology magazine " (J.Cell Biol.) 1988,106:1281-1288), and in conjunction with the monoclonal antibody of those fraction otherwise can not allow to have promoted in the substrate of axon growth the sensation of being cultivated and growth (the Caroni ﹠amp of sympathetic neuron; Schwab, " neuron " (Neuron) 1988,1:85-96).Prove also that with these identical antibody researchs in the pathological changes animal functional restoration can obtain (people such as Bregman, " nature " (Nature) 1995,378:498-501 by the function of the sealing inhibition molecule relevant with myelin; Schnell ﹠amp; Schwab, " nature " (Nature) 1990,343:269-272).Obtained more sound regenerative response people such as (, 1999) Huang in complete myelin mice immunized, this has further proved the recovery of CNS and has repaired and can be enhanced in vivo by the sealing inhibitive factor.

The molecule in known at least three kinds of myelin source is effective axon growth inhibitor: the myelin associated glycoprotein, also be referred to as " MAG " (by people such as McKerracher, " neuron " (Neuron) 1994, people such as 13:805-811 and Mukhopadhyay, " neuron " (Neuron) 1994,13:757-767 describes); Nogo-A (referring to people such as Chen, " nature " (Nature) 2000,403:434-439; People such as GrandPre, " nature " (Nature) 2000, people such as 403:439-444 and Prinjha, " nature " (Nature) 2000,403:383-384) and the oligodendroglia myelin glycoprotein (people such as Wang, " nature " (Nature) 2002,417:941-944).Nogo receptor (also being referred to as " NgR "), ganglioside GT1b and p75 neurotrophin receptor (also being referred to as " p7SNTR " or " p75NTR ") relate to mediation these three kinds reactions that suppress molecule at all.Particularly, the inhibition activity of whole three inhibitor MAG, Nogo-A and oligodendroglia glycoprotein necessary (people such as Domeniconi, 2002 are it is said in the combination of NgR; People such as Liu, 2002; People such as Wang, 2002b).Yet MAG also can be directly in conjunction with GT1b receptor (Vyas﹠amp; Schnaar, " biochemistry " (Biochimie) 2001,83:677-682).In addition, induce the inhibitory reaction that the antibody capable simulation of GT1b receptor clustering produces by MAG (referring to people such as Vinson, " journal of biological chemistry " (J.Biol.Chem.) 2001,276:20280-20285; With people such as Vyas, " NAS's progress " (Proc.Natl.Acad.Sci.U.S.A.) 2002,99:8412-8417).

The p75 receptor is can be in conjunction with the signal conduction composition of the polymer receptor complex of whole three kinds of myelin receptors.Referring to people such as Domeniconi, " neuron " (Neuron) 2002,35:283-290; People such as Liu, " science " (Science) 2002,297:1190-1993; People such as Wang, " nature " (Nature) 2002,417:941-944.GT1b and p75 ^NTRInteraction between the receptor is in the news (people such as Yamashita, 2002), with NgR and p75 ^NTRThe interaction that has between the receptor the same (referring to people such as Wang, people such as 2002a and Wong, 2002).These and p75 ^NTRInteraction to be considered to for suppressing the transmission that signal (as being selected from MAG, Nogo-A and/or oligodendroglia glycoprotein) passes cell membrane be important.For example, the interaction between the cell of MAG or Nogo-A peptide and expression NgR has improved p75 ^NTRWith the combination of Rho-GDI, and induced release (the Yamashita ﹠amp of RhoA from this complex; Tohyma, " Nature Neuroscience " (Nat.Neurosci.) 2003,6:461-471).This step is primary for the activation of RhoA and the inhibition of growth (Id), and myelinic the inhibitions activity in the neuron that the inhibition of RhoA and/or Rho kinases (downstream effect of RhoA) has been avoided for example cultivating effectively is (referring to for example, people such as Dergham, " Journal of Neuroscience " (J.Neurosci.) 2002,22:6570-6577; People such as Fournier, " Journal of Neuroscience) " (J.Neurosci.) 2003,23:1416-1423; With people such as Lehmann, " Journal of Neuroscience " (J.Neurosci.) 1999,19:7537-7547).

As noted above, different neurotrophin (as NGF, BDNF, NT-3 and NT-4/5) has remarkable influence really to the growth of neuronic existence between the period of development and aixs cylinder.Think that recently neurotrophin and inhibition molecule (for example, MAG, Nogo-A and oligodendroglia glycoprotein) may be to p75 ^NTRReceptor is combined with opposite effect (referring to Yamashita ﹠amp to Rho-GDI's; Tohyama, " Nature Neuroscience " (Nat.Neurosci) 2003,6:461-467).Yet, use neurotrophin to promote sound, long-range axon regeneration to be still impossible at this moment.This is considered to is because neurotrophin can not effectively resist those inhibition signals as indicated above at least in part.For example, it is active to use the neuron of handling cultivation such as the neurotrophin of NGF, BDNF or GDNF (neuroglia derived neurotrophic factor) not resist myelinic inhibition usually, unless these neurons before the contact inhibition signal by contact neurotrophin several hours at first by " sensitization " (people such as Cai, Neuron 1999,22:89-101).Yet this sensitization effect is limited, expend time in, use trouble, and impracticable in using in clinical and other body.And (as noted above), neurotrophin self is a macromole, its therapeutic send constituted sizable, may be unsurmountable technological challenge.In addition, because neurotrophin is attached on the inhibitor complexes by itself and the interaction of p75 receptor, so they promote that regenerated ability energy is impaired.Can not activate the Trk receptor in conjunction with the neurotrophin of p75 receptor overcomes and suppresses the signal conduction and promote neure growth.

Therefore, exist in addition and suppress the needs of signal the chemical compound of the effect of neure growth and recovery to effectively regulating, comprise effective adjusting for example those by MAG, Nogo-A, oligodendroglia glycoprotein, NgR, GT1b, p75 ^NTRAnd/or the chemical compound of the effect of the inhibition signal that produced of the downstream effect thing of these signaling molecules.Especially, suppress signal to effectively resisting these, and/or the chemical compound of stimulating neuronal growth and recovery exists needs.To regulating process and the method (comprising Therapeutic Method) that these suppress signal effects, and particularly also exist needs to resisting these process and methods that suppress signals and/or stimulating neuronal growth and recovery.

Description of the invention is explained in the only confession of quoting and/or discuss to list of references in this part and whole description, and is not to admit that any such list of references is a prior art of the present invention described here.

4. summary of the invention

The present invention provides to the solution of small part the problems referred to above by active chemical compound and the preparation thereof that adjusting (as strengthening or inhibition) Trk receptor such as TrkA, TrkB or TrkC mediation are provided.For example, in one embodiment, the invention provides is the Trk antagonist, and thereby suppresses the active chemical compound of Trk mediation.In other embodiments, the invention provides is the Trk agonist, and thereby improves or strengthen the active chemical compound of Trk mediation.

As noted above, Trk receptor and part thereof (being neurotrophin such as NGF, BDNF, NT-3, NT-4, NT-5 and NT-4/5) are relevant with reparation with central nervous system's (CNS) growth.Thereby Trk regulator chemical compound of the present invention can be used to regulate such process, comprises that neuronic growth and existence, axon growth, neurite grow and synaptic plasticity.Therefore, on the one hand, the invention provides and use Trk regulator chemical compound of the present invention to regulate the method (comprising Therapeutic Method) of (strengthening for instance, or inhibition) these processes.

In a special embodiment, the invention provides and regulate the receptor-mediated active cyclic peptide compounds of Trk.These cyclic peptide preferably comprise aminoacid sequence: Arg-Gly-Glu in its ring.In more special embodiment, this cyclic peptide comprises structural formula:

(formula I)

In formula I, composition Y ₁And Y ₂Be independently selected from and have the Y of being formed on ₁And Y ₂Between the aminoacid of covalent bond.Component X ₁And X ₂Choose wantonly, and if present, the aminoacid sequence that is independently selected from aminoacid or connects by peptide bond.Preferred X ₁And/or X ₂Length respectively 0 and about 10 aminoacid between, more preferably length is approximately 1,2,3,4 or 5 aminoacid.In addition, the also preferred X that so selects ₁And X ₂Thereby the magnitude range of cyclic peptide ring is about 5 to about 15 amino acid whose length, and more preferably length is about 5-10 aminoacid.

In special embodiment, the present invention further provides cyclic peptide with following structural formula:

Or

Wherein, Y ₁And Y ₂Described in following formula I.The particularly preferred cyclic peptide of the present invention is to comprise aminoacid sequence: CSRRGEC(SEQ ID NO:1), N-Ac- CSRRGEC-NH ₂(SEQ ID NO:2), CARRGEC(SEQ ID NO:3), N-Ac-CARRGEC-NH ₂(SEQ ID NO:4), CFHRGEC(SEQ ID NO:5), N-Ac- CFHRGEC-NH ₂(SEQ ID NO:6), CSHRGEC(SEQ ID NO:7), N-Ac- CFHRGE-NH ₂(SEQ ID NO:8), CRGEC(SEQ ID NO:9), N-Ac- CRGEC-NH ₂(SEQ ID NO:10), N-Ac- KRGED-NH ₂(SEQ ID NO:11), H-C (O)- CRGEC-NH ₂(SEQ ID NO:12), CH ₃-SO ₂-NH- CRGEC-NH ₂(SEQ ID NO:13), N-Ac- CRGEC-Y-NH ₂(SEQ ID NO:14), H-C (O)- CRGEC-Y-NH ₂(SEQ ID NO:15) and CH ₃-SO ₂-NH- CRGEC-Y-NH ₂The cyclic peptide of (SEQ ID NO:16), (wherein the line of each aminoacid sequence partly refers in the peptide by the part of cyclisation).

In the top structural formula and sequence, preferred cyclic peptide is the Trk antagonist.Yet in other embodiments, it is the cyclic peptide of Trk agonist that the present invention also provides.These cyclic peptide preferably have following structural formula:

(formula II)

In above-mentioned formula II, composition Y ₁And Y ₂Be independently selected from and have the Y of being formed on ₁And Y ₂Between the aminoacid of covalent bond.Composition Z ₁, Z ₂And Z ₀Choose wantonly, and if present, the aminoacid sequence that is independently selected from aminoacid or connects by peptide bond.Preferred Z ₁, Z ₂And/or Z ₀Length be respectively only about 10 aminoacid, more preferably length has only about 1,2,3,4,5 or 10 aminoacid.In addition, Z ₁, Z ₂And/or Z ₀Length preferred thereby so to select the magnitude range of cyclic peptide ring be 10-50 amino acid whose length, more preferably length is about 10-25 aminoacid or about 15-20 aminoacid.In particularly preferred embodiments, selection component Z like this ₁, Z ₂And/or Z ₀Thereby the series connection Arg-Gly-Glu sequence among the formula I is taked a kind of their adjacent one another are and antiparallel conformations.

In preferred embodiments, the invention provides cyclic peptide with following structural formula according to formula II:

Composition Y wherein ₁And Y ₂The same as described in to formula II.Particularly preferred also is that the peptide according to formula II of a part of the present invention is to comprise aminoacid sequence CSRRGELAASRRGELC(SEQ IDNO:17) and N-Ac- CSRRGELAASRRGELC-NH ₂The cyclic peptide of (SEQ ID NO:18), (wherein the line of each aminoacid sequence partly refers in the peptide by the part of cyclisation).

According to the present invention, the cyclic peptide that is provided comprises the D aminoacid sequence in its ring ring:

dGlu-Gly-dArg。

Wherein this cyclic peptide is regulated the receptor-mediated activity of Trk.The receptor-mediated active cyclic peptide of adjusting Trk that preferably comprises the D aminoacid sequence is c[dLdEGdRdRdSdLdEGdRdRdS] (SEQ ID NO:40) (wherein the parenthesis part of this aminoacid sequence refers to by the part of the peptide of peptide bond cyclisation) and Ac- DCdLdEGdRdRdSdAdAdLdEGdRdRdSdC-NH ₂(SEQ IDNO:41).

In another embodiment preferred, the invention provides have aminoacid sequence c[SRRGELSRRGEL] cyclic peptide of (SEQ ID NO:39).

Except cyclic peptide, the present invention also provides other to regulate the method that the receptor-mediated activity of Trk maybe may be regulated this active chemical compound (i.e. " candidate compound ").These methods comprise that the three dimensional structure with the three dimensional structure of candidate compound and cyclic peptide of the present invention compares.Similarity between candidate compound structure and the cyclic peptide structures shows the receptor-mediated active ability of candidate compound adjusting Trk.Therefore, have with the three dimensional structure of cyclic peptide basically the candidate compound of analog structure be likely and regulate the receptor-mediated active chemical compound of Trk.

Said method is applicable in theory identifies the receptor-mediated active peptide mimics chemical compound of adjusting Trk.Correspondingly, the invention provides is the peptide mimics chemical compound of Trk regulator, and these chemical compounds are considered to another aspect of the present invention.Especially, peptide mimics chemical compound of the present invention is to have with cyclic peptide of the present invention (promptly to regulate the active cyclic peptide of Trk mediation, and comprise aminoacid sequence in its ring ring: the chemical compound of the three dimensional structure that three dimensional structure Arg-Gly-Glu) is similar basically.

The present invention provides such method (comprising Therapeutic Method) in addition, and this method is to use cyclic peptide and peptide mimics chemical compound to regulate the activity of Trk mediation.In such embodiment, the invention provides the active method that suppresses the Trk mediation.This method comprises cell (body in or external) is contacted with cyclic peptide active of the present invention or the peptide mimics chemical compound that a certain amount of inhibition Trk mediates.With the cyclic peptide of cells contacting or the amount of peptide mimics chemical compound should be effectively to suppress the receptor-mediated active amount of Trk.

In another embodiment, the invention provides the active method that strengthens the Trk mediation.This method comprises cell (body in or external) is contacted with cyclic peptide active of the present invention or the peptide mimics chemical compound that a certain amount of enhancing Trk mediates.With the cyclic peptide of cells contacting or the amount of peptide mimics chemical compound should be effectively to strengthen the receptor-mediated active amount of Trk.

Can be comprised by the active embodiment of the Trk mediation of these methods adjustings (enhancing or inhibition for instance): neuronic growth and survival, axon growth, neurite grow other process with synaptic plasticity and central nervous system (CNS) growth and/or reparation.Therefore, the present invention provides the growth that promotes individual central nervous system and the method for reparation in addition.These methods comprise and give the individual a certain amount of cyclic peptide active of the present invention or peptide mimics chemical compound that can strengthen the Trk mediation.The cyclic peptide that gives or the amount of peptide mimics should be effectively to promote CNS growth or the amount of repairing.

The present invention provides the method for the reaction of using Trk agonist and antagonist to regulate to suppress the CNS growth and repairing in addition, comprises that common inhibition such as neure growth, neuronal survival, axon growth, neurite grow the reaction with the process of synaptic plasticity.Especially, Trk agonist of the present invention and antagonist can be used to regulate inhibitive factor and/or by inhibition signal that these factors produced.Example comprises the factor relevant with myelin, comprises myelin associated glycoprotein (MAG), Nogo-A and oligodendroglia myelin glycoprotein.Generally speaking, the invention provides and use Trk agonist and/or antagonist to regulate method by the CNS inhibitor reaction of signal cascade mediation, described signal cascade has the component of the adjusting of one or more one or more factors that self are subjected to participating in the conduction of Trk receptor signal.These comprise, for example, and as the component of the Rho that regulated by protein kinase A (PKA) and/or phosphoinositide-3 kinases (P13K).Therefore, in preferred embodiments, the invention provides by Trk agonist of the present invention (cyclic peptide or peptide mimics for instance) and contact the method that reduces this " CNS inhibitor " reaction cell and the amount that effectively reduces the reaction of CNS inhibitor.The present invention also provides by the amount with the reaction of effective reduction CNS inhibitor and has given the method that individual a certain amount of Trk agonist of the present invention (cyclic peptide or peptide mimics for instance) reduces individual CNS inhibitor reaction.

Again in other embodiments, the invention provides the pharmaceutical composition that can be used for the treatment of method, for example above-described those (Therapeutic Method).These pharmaceutical compositions comprise a certain amount of cyclic peptide of the present invention or peptide mimics chemical compound, together with one or more pharmaceutically and/or the physiology go up acceptable carrier, diluent or excipient.

In addition, the present invention is based on a kind of like this discovery, promptly disturb neurotrophin that the reagent of p75 receptors bind is promoted the CNS neure growth in suppressing environment.

Trk receptor and part thereof (being neurotrophin such as NGF, BDNF, NT-3, NT-4 and NT-5) are with the neuronic growth of CNS and repair relevant.Thereby, when neurotrophin in conjunction with and when activating the Trk receptor, the Trk triggered promote the signal transduction cascade of neure growth.Yet in conjunction with neurotrophin, and when the p75 receptors bind is on inhibitor complexes, this interaction has damaged the ability that neurotrophin promotes the CNS neure growth to the p75 receptor with low-affinity.The invention provides use can disturb neurotrophin to promote the method for CNS neure growth in conjunction with the p75 receptor-binding agents of p75 receptor.

According to the present invention, the method that promotes the CNS neure growth in suppressing environment is provided, this method comprises the p75 receptor-binding agents that gives the individual treatment effective dose.In one embodiment, the p75 receptor-binding agents comprises neurotrophin binding motif or its peptide mimics.In an especially preferred embodiment, the p75 receptor-binding agents is included in cyclic peptide or the peptide mimics that comprises aminoacid sequence Thr-Asp-Ile-Lys-Gly-Lys-Glu (TDIKGKE) (SEQ ID NO:42) in its ring ring.Preferred p75 receptor-binding agents is N-Ac- CTDIKGKEC-NH ₂(SEQ ID NO:43).Individuality is mammal preferably, is more preferably the people.

The invention provides the method that promotes the CNS neure growth in suppressing environment, this method comprises the p75 receptor-binding agents association neurone nutrient protein that gives the individual treatment effective dose.In one embodiment, neurotrophin is selected from NGF, BDNF, NT-3, NT-4 and NT-5.In further embodiment, to give p75 receptor-binding agents greater than neurotrophin about 10 to about 100 times amount.In one aspect of the invention, the p75 receptor-binding agents is a neurotrophin, and it disturbs other different neurotrophin in conjunction with the p75 receptor, but does not disturb this other different neurotrophin in conjunction with the Trk receptor that is expressed on the injured neurons.One special aspect, the p75 receptor-binding agents is NGF, and neurotrophin is BDNF, wherein NGF is to give to about 100 times amount for about 10 times greater than BDNF.Individuality is mammal preferably, is more preferably the people.

5. brief description of drawings

Fig. 1 has shown the aminoacid sequence comparison of NTs BDNF (SEQ ID NO:19), NT4 (SEQ ID NO:20), NT3 (SEQ ID NO:21) and NGF (SEQ ID NO:22).Ripe peptide chain represents with bold-type letter, and underlined be the RGE motif.

Fig. 2 A-2C has shown the comparison of the full length amino acid sequence of people TrkA (SEQ ID NO:23), TrkB (SEQ ID NO:24) and TrkC (SEQ ID NO:25) receptor.The consensus sequence of these receptors is by on the frame, and the border in receptor different structure territory marks with vertical line.Also referring to people's such as Presta U.S. Patent No. 5,844,092.

Fig. 3 A-3B has illustrated the representative backbone modifications that may be present in the peptide mimics.Also referring to Fig. 4 A and 4B among the WO 01/53331.

Fig. 4 has illustrated the representativeness that may be incorporated in the peptide mimics not common amino acid and dipeptides succedaneum.Also referring to the Fig. 5 among the WO 01/53331.

Fig. 5 has illustrated the representative secondary structure analogies that may be incorporated in the peptide mimics.Also referring to the Fig. 6 among the WO 01/53331.

Fig. 6 A-6C illustrates and analyzes the NT/Trk crystal structure with the part range of linearity of evaluation with the Trk acceptor interaction.Fig. 6 A has shown with two nearly film Ig domains from the TrkB receptor and (has been expressed as chain b ₁And b ₂) NT-4 dimer in the formed complex (is expressed as chain a ₁And a ₂) crystal structure band representation (by people such as Banfield report, Structure (Camb) 2001,9:1191-1199).Fig. 6 B has shown research this crystal structure (solid line) and NGF/TrkA crystal structure, and (by people such as Wiesman report, Nature 1999, and 401:184-188) (dotted line) makes a to find ₁And b ₂The result of the linear peptides sequence (LIPs) that contacts between the chain.Fig. 6 C has shown that research NGF (dotted line) and NT-4 (solid line) crystal structure can make a to find ₁And b ₁The result of the LIPs that contacts between the chain.

Fig. 7 A-7D shown fix and GAP-43 dyeing before, in the presence of different peptides, in control medium or replenished on the monolayer 3T3 cell in the culture medium of NT-4 or BDNF and cultivated cerebellar neuron after 18 hours, the experimental data that is obtained.Under every kind of condition of culture, record the average length of long neurite from the neuron between about 100-120.Fig. 7 A has shown the NT-4 of test variable concentrations and the experimental data that influence obtained that BDNF grows neurite.Fig. 7 B has shown in control medium and has contained in the culture medium of 5ng/ml BDNF or 5ng/ml NT-4 as shown in the figure that test increases cyclic peptide N-Ac- CRGEC-NH ₂The experimental data of the influence that concentration produced.Fig. 7 C has shown the cyclic peptide N-Ac-of test derived from NT-4 CSRRGEC-NH ₂(SEQ IDNO:26), derived from the cyclic peptide N-Ac-of NT-3 CSHRGEC-NH ₂With cyclic peptide N-Ac-derived from NGF CFHRGEC-NH ₂Experimental data to the influence of the cerebellar neuron cultivated with 5ng/ml BDNF.Fig. 7 D has shown and identical experimental data shown in Fig. 7 C, but cerebellar neuron is wherein cultivated with 5ng/ml NT-4.

Fig. 8 has illustrated at control medium or additional NT-4, BDNF, FGF2 (all are 5ng/ml), has replenished CG1 receptor stimulating agent WIN55, on the monolayer 3T3 cell in the culture medium of 2122-2 (0.2 μ M) or the experimental result of cultivating cerebellar neuron on the monolayer 3T3 of the N-of its cell surface expression transfection cadherin (NCAD) cell.There are or do not exist (a) 440 μ M cyclic peptide N-Ac- CRGEC-NH ₂(b) 125 μ M cyclic peptide N-Ac- CSRRGEC-NH ₂(c) 125 μ M linear peptides N-Ac-SRRGELA-NH ₂Experimentize and data are mapped under the situation of (SEQ ID NO:27).

Fig. 9 A-C has shown B _AGThe model structure of peptide.Fig. 9 A has shown the natural structure from the dimeric monomeric SRRGELA motif of the NT-4 in the NT-4/TrkB crystal structure.Be shown among Fig. 9 C from the natural structure of the monomeric ASRRGEL of gametophyte NT-4 (the SEQ ID NO:28) motif of this crystal structure.Mixed the B of these " series connection-repetition " motifs _AGPeptide N-Ac- CSRRGELAASRRGELC-NH ₂Model structure be shown among Fig. 9 B.

Figure 10 A-10B has shown at the B that has replenished the finite concentration scope _AGPeptide N-Ac- CSRRGELAASRRGELC-NH ₂Culture medium in cultivate cerebellar neuron neurite grow experimental result.Figure 10 A has shown the meansigma methods of the absolute neurite lengths that 100-120 neuron of sampling records from single experiment.Figure 10 B has shown relatively B _AGThe effect of peptide (6 μ M) and block diagram at the reaction of the growth promoter of NT-4, BDNF shown in fixed comprising and FGF2 (all are 5ng/ml).

Figure 11 has shown from control medium or (a) the 6 μ M BAG peptide N-Ac-shown in replenished CSRRGELAASRRGELC-NH ₂(b) 125 μ M TrkB antagonist peptide N-Ac- CSSRGEC-NH ₂(SEQ ID NO:29); (c) 100nM Trk specificity tyrosine kinase inhibitor K252a; Or the TrkB antagonist peptide N-Ac-SRRGELA-NH of 125 μ M linear forms ₂Culture medium in cultivate the neurite that cerebellar neuron obtains and grow experimental result.

Figure 12 has shown that the neurite of the effect of describing the different reagent of test grows the bar diagram of experimental result.Particularly, the monolayer in having replenished the culture medium of soluble M AG-Fc fusion constructs body that final concentration is 0,5 or 25 μ g/ml (shown in each bar line below among the figure) is expressed on the 3T3 cell of N-cadherin and is cultivated cerebellar neuron.In control medium (just, only having replenished the culture medium of MAG-Fc) with additionally replenished Rho inhibitors of kinases Y27623 (final concentration 10 μ M), B _AGExperimentize in the culture medium of polypeptide (final concentration 6 μ M) or BDNF (final concentration 5ng/ml).Fix and GAP-43 dyeing before keep and cultivated 22 hours.Under every kind of condition of culture, from measuring, the neuron between about 100-120 records the average length of long neurite.Each post of chart has been described the amalgamation result from many independent experiments (shown in the post top), and the bar line is represented standard error of mean (SEM).

Figure 13 has illustrated B in the neuron of cultivating _AGPolypeptide is to the dose-effect curve of MAG-Fc reaction.Particularly, each data point is represented, when being incubated at the B that has replenished final concentration shown in MAG-Fc (25 μ g/ml final concentration) and the transverse axis _AGWhen monolayer in the culture medium of polypeptide is expressed on the 3T3 cell of N-cadherin, the average length that records from about 120-150 neuron.The data that each some expression obtains from single representative test, the bar line on each point is represented SEM.

Figure 14 has shown a bar diagram, and its description is incubated at the monolayer 3T3 cell of not expressing the N-cadherin and has replenished test b in the culture medium of MAG-Fc (as shown in the figure) of 0 or 25 μ g/ml final concentrations _AGPolypeptide and BDNF grow the experimental result of influence to neurite in the cerebellar neuron.Experiment is in control medium (just, only having replenished the culture medium of MAG-Fc) and additionally replenished B _AGCarry out in the culture medium of polypeptide (6 μ M final concentration) or NDNF (5ng/ml final concentration).Fix and GAP-43 dyeing before keep and cultivated 22 hours.Under every kind of condition of culture, record the average length of long neurite from the neuron measurement between about 100-120.Each post of chart is represented the amalgamation result from three independent trialss, and the bar line is represented standard error of mean (SEM).

Figure 15 shows a bar diagram, it is described test b AG polypeptide and expresses the experimental result of the influence that neurite grows in the cerebellar neuron on the 3T3 cell of N-cadherin to being incubated at monolayer, and these 3T3 cells are cultured in: the control medium that (C) does not have fill-in; (1) replenished the culture medium of the monoclonal antibody (20 μ g/ml final concentration) of GT1b; Or (2) have replenished GT1b antibody (20 μ g/ml final concentration) and B _AGIn both culture medium of polypeptide (6 μ M final concentration).Fix and GAP-43 dyeing before keep and cultivated 22 hours.Under every kind of condition of culture, record the average length of long neurite from the neuron between about 100-120.Each post among the figure represents that the bar line on each post is represented SEM from the amalgamation result of the independent trials between 7 and 10.

Figure 16 has shown a bar diagram, and it describes the different reagent of test to being incubated at the control medium (post C) that do not have fill-in or with 75 ^NTRThe pretreated culture medium of antibody (post 1-4) in monolayer express the experimental result of the influence that neurite grows in the cerebellar neuron on the 3T3 cell of N-cadherin.The control medium that does not have fill-in in (1); (2) Rho inhibitors of kinases Y27632 (10 μ M final concentration); (3) B _AGPolypeptide (6 μ M final concentration); Or after handling in (4) BDNF neurotrophin (5ng/ml final concentration), cultivate these neurons through antibody treatment.Fix and GAP-43 dyeing before keep and cultivated 22 hours.Under every kind of condition of culture, record the average length of long neurite from the neuron between about 100-120.Each post among the figure is represented the amalgamation result from the independent experiment of number shown in the top, and the bar line on each post is represented SEM.

Figure 17 has shown a bar diagram, and it describes the different inhibitors of kinases of test to being incubated at the experimental result of the influence that neurite grows in the cerebellar neuron under the different condition.Especially, to be cultured in and to have replenished final concentration be the B of 6 μ M to cerebellar neuron _AGPolypeptide (blank bar line), final concentration are on the monolayer 3T3 cell in the culture medium of the BDNF neurotrophin (striped bar line) of 5ng/ml or the FGF2 that final concentration is 5ng/ml (black bar line).For testing the effect of different reagent, experiment as shown in the figure, use the control medium that does not contain extra fill-in, or use the culture medium of additionally having replenished K252a (100nM final concentration), PKA inhibitor (final concentration is the KT5720 of 200nM, or final concentration is the H-89 of 400nM) or PI3K inhibitor (final concentration is respectively wortmannin or the Ly294002 of 10 μ M) to carry out.Fix and GAP-43 dyeing before keep and cultivated 18 hours, and under every kind of condition of culture, the neuron between about 100-120 records the average length of long neurite.Data to the result that obtains with every kind of PKA inhibitor and every kind of PI3K inhibitor (it produces identical result) are gathered.Each post among the figure is represented the amalgamation result from least 3 independent experiments, and the lines on each post are represented SEM.

What Figure 18 A-18B showed is chart, and they have been described test and have been dissolved in distilled water (dH to being incubated at bag ₂The mixture of 17 μ g/ml polylysines, goat-anti-human IgG (Fc-is specific) and fibronectin 0) (both is dissolved among the DMEM, 10 μ g/ml) and the experimental result that is dissolved in the influence that neurite grows in the cerebellar neuron in " the inhibition environment " in the hole of 0.25 μ g/ml MAG-Fc of DMEM/10%FCS.Fix and GAP-43 dyeing before keep and cultivated 27 hours.Figure 18 A has shown at hriB _AG2, hB _AG2Or riB _AGThere is the dose-effect curve of the average neurite lengths of the cerebellar neuron of growth down.Figure 18 B shows a bar diagram, and it is described in BDNF, B _AG, hriB _AG2, hB _AG2Or riB _AGThere is the average neurite lengths of the cerebellar neuron of growth down.

Figure 19 has shown a bar diagram, and its neurite of describing the effect of different reagent in the test inhibition environment grows experimental result.Particularly, cerebellar neuron is incubated at the monolayer that has replenished in the culture medium of soluble M AG-Fc fusion constructs that final concentration is 25 μ g/ml and expresses on the 3T3 cell of N-cadherin.Cultivation further replenishes BDNF (1ng/ml), NGF (10ng/ml or 100ng/ml), BDNF (1ng/ml) unites NGF (10ng/ml or 100ng/ml), and 100 μ g/ml NGF encircle 1 binding motif (N-Ac- CTDIKGKEC-NH ₂) the constraint monomer or the NGF of (SEQ ID NO:43) encircle 1 peptide (100 μ g/ml) Associated with BDNF (1ng/ml).Fix and GAP-43 dyeing before keep and cultivated 23 hours.Under every kind of condition of culture, record the average length of long neurite from the neuronic measurement between about 100-120.Each post of chart is described the amalgamation result from many independent experiments (shown in the post top), and the bar line is represented the standard units of the error of the mean (SEM).

6. summary of the invention

As noted above, the invention provides the compound that comprises peptide and peptide mimics, their regulate the activity that (increase for instance, or reduce) mediated by Trk-acceptor such as TrkA, TrkB and TrkC. These compounds are commonly referred to as Trk-receptor modulator compounds or " Trk conditioning agent " at this.

Trk conditioning agent of the present invention can be used for, for instance, regulate such as neure growth and survival, axon growth, neural process grow, the process of synaptic plasticity and other is at least partially by the receptor-mediated process of Trk-. These purposes comprise and can (for example patient or other individuality) participate in regulating the methods for the treatment of that central nervous system is grown and repaired in external (for instance, in cell culture) or body. Therefore, Trk conditioning agent of the present invention only gives some instances in treatment, is effective in the disease as apoplexy, Alzheimer's, Parkinson's, head and spinal cord injury and this class of epilepsy.

The applicant has found that the interaction of a key between Trk acceptor and their neurotrophin part is that conservative short-term sequence motifs-Arg-Gly-Glu " RGE " of single amino acids coded representation (namely with) by one section three amino acid residue occurs, and it is terminal that this motif sees the N-of ripe neurotrophin amino acid sequence. The RGE motif is present in all neurotrophins, and on being attached to the Trk acceptor time, is present in the place that is considered to sealing ring (tight loop) as half spiral.

The applicant finds that also the suitable constraint peptide (for example, cyclic peptide) with little linear RGE motif has the Structural superposition of height with natural NT structure, and can be as the antagonist performance function of Trk acceptor. Similarly, have the overlapping peptide mimics compound of attach structure with these constraint RGE peptides and also expect the Structural superposition that has height with natural NT structure, and thereby also can be as Trk receptor antagonist performance function.

As noted above, the RGE motif is guarded in all neurotrophins, and with the interaction of this motif be important for the combination of those neurotrophins and their Trk acceptors separately. Therefore, comprise the constraint peptide of RGE motif and the antagonist that peptide mimics can be used as the numerous Trk acceptor that comprises TrkA, TrkB and TrkC. Yet by selecting the preferential flanking amino acid sequence in conjunction with expectation Trk acceptor from the NT part, Trk conditioning agent of the present invention also can the specific Trk acceptor of target. In preferred Trk agonist compounds (the Trk conditioning agent compound that namely suppresses the receptor-mediated activity of Trk), the length range of these flank residues is about 0 to 10 amino acid residue only preferably, and wherein size is particularly preferred at the about amino acid residue between 2-5 or the 2-3. In addition, the size of cyclic peptide ring (or corresponding peptide mimics structure) preferably only has about 4 to 15 amino acid residues, and wherein size is particularly preferred from about amino acid residue of 5 to 10.

The applicant also determined in the dimeric crystal structure of the NT in the compound of Trk receptors bind domain, RGE motif self arranged anti-parallel in the NT dimer. That is to say that the intramolecular RGE spiral of second NT in the intramolecular RGE spiral of first NT and this dimer forms a line, and is antiparallel orientations. Referring to, Fig. 6 A-6C particularly. And the applicant finds, the peptide of the RGE motif that repeats when series connection or peptide mimics are during by suitable constraint (as in cyclic peptide or peptide mimics), and it takes identical arranged anti-parallel conformation, and has the Structural superposition of height with natural NT structure. RGE cyclic peptide and the peptide mimics of this " series connection-repeat " can be surprisingly as Trk receptor stimulating agent (being that they can strengthen the receptor-mediated activity of Trk) performance functions. Thereby these compounds are also within Trk conditioning agent compound of the present invention.

As the RGE antagonist that preamble is described, the constraint peptide of the RGE motif that comprising connects repeats and the activator that peptide mimics can be used as the numerous Trk acceptor that comprises TrkA, TrkB and TrkC. Yet for example, by selecting the preferential flanking amino acid sequence in conjunction with expectation Trk acceptor from the NT part, these compounds also can the specific Trk acceptor of target. In preferred Trk agonist compound (namely strengthening the Trk conditioning agent compound of the receptor-mediated activity of Trk), the length range of these flank residues is about 0 to 10 amino acid residue only preferably, wherein more preferred at 2-5 or 2-3 of size.

Randomly, cyclic peptide and peptide mimics that series connection of the present invention repeats can comprise extra amino acid residue, and these residues are between the RGE motif that two series connection repeat. Therefore, these extra amino acid residues are brought into play function as " spacerarm " part by this way, two RGE motifs are linked together, thus two RGE motifs take with natural NT structure in the RGE motif have the overlapping arranged anti-parallel conformation of attach structure. The spacerarm amino acid residue really personal part unimportant, and their identity is may or may be not corresponding with the identity of amino acid residue of RGE motif in the specific neurotrophin of side joint. Preferably, the cyclic peptide of series connection repetition or the spacerarm motif (if present) in the peptide mimics are short; For instance, length is no longer than 5 amino acid residues, and wherein the spacerarm motif of length between about 0-3 amino acid residue is more preferred. The length of particularly preferred spacerarm motif only has about 1 or 2 amino acid residue.

In addition, total size of cyclic peptide of these " series connection-repeat " and peptide mimics is typically Trk antagonist cyclic peptide of the present invention or peptide mimics about 2 times. Therefore, it is long that the preferred size of peptide ring (or structure of corresponding peptide mimics) is preferably about 8 to 30 amino acid residues, and wherein size is that about 10 to 20 amino acid residues are long particularly preferably.

The cyclic peptide that preferably comprises RGE motif and/or its series connection repetition is described in hereinafter 6.1 joints. Next 6.2 joints have been described conventional experimental technique, by these methods, those skilled in the art can, for instance, by X-radiocrystallgraphy or NMR spectroscopy, measure three-dimensional " pharmacophore " structure of these and other cyclic peptide, and the method that designs suitable peptide mimics compound with these pharmacophore structures is provided in hereinafter 6.3 joints together with exemplary peptide mimics modification. Further modification to Trk conditioning agent compound of the present invention is described in 6.4 joints, comprises pharmaceutical preparation and medical usage. 6.5 joint has been described routine test, by these tests, those skilled in the art can verify that the Trk conditioning agent of compound such as peptide mimics compound is active. Then 6.6 joints have been described in the method for the activity of regulating the Trk mediation such as neuronal survival, axon growth and synaptic plasticity, the preferred exemplary purposes of these compounds. The preparation (comprising pharmaceutical preparation) that is specially adapted to the Trk conditioning agent of these purposes is provided in 6.7 joints. At the 7th joint, this specification finishes with a series of embodiment, has showed some exemplary embodiment of the present invention.

The present invention also provides the method that is used for to individual administration p75 receptor-binding agents, and wherein the p75 receptor-binding agents disturbs neurotrophin to being combined in the combination of the p75 acceptor in the inhibitor complexes, and therefore promotes neurotrophin in conjunction with the Trk acceptor. These methods are in treatment CNS neuronal damage or injured situation, and for example, the disease of a class is useful as apoplexy, Alzheimer's, Parkinson's, the injury of traumatic head and spinal cord injury.

The present invention partly is based on such discovery, namely is designed to disturb neurotrophin that the reagent of the combination that is combined in the p75 acceptor in the inhibitor complexes is promoted that neurotrophin-mediated CNS neural process grows. Described in following embodiment one joint, the cerebellar neuron of the rat that use is cultivated in suppressing environment has carried out neural process and has grown test. This inhibition culture media supplemented has NGF, NGF Associated with BDNF, NGF the first coupling collar (N-Ac-CTDIKGKEC-NH ₂) constraint monomer or the described NGF of (SEQ ID NO:43) encircle 1 peptide Associated with BDNF. Behind the maintain 23 hours, fixing, carry out GAP-43 dyeing. Then under every kind of condition of culture, record the average length of long neural process from 100-120 neuron. Research finds that independent NGF and independent constraint monomer can not promote that neural process grows, but the BDNF that NGF and BDNF unite with the ratio of 10: 1 and 100: 1 and the NGF of 100 μ g/ml encircles 1 peptide and 1ng/ml unites and promoted neural process to grow. These find to show that administering drug combinations p75 receptor-binding agents (its for not in conjunction with the neurotrophin of the Trk acceptor of expressing) neurotrophin (it in conjunction with the Trk acceptor that be expressed in injured neurons on) different with other caused the CNS neuron to be grown in the inhibition environment. At this, the neurotrophin (being the p75 receptor-binding agents) of the Trk acceptor of combination expression is not to arrive about 100 times amount administration greater than the neurotrophin (being non-p75 receptor-binding agents) about 10 in conjunction with the Trk acceptor of expressing. These results also show the constraint monomer of administration NGF the first coupling collar, a kind of non--neurotrophin p75 receptor-binding agents, promoted the neuronic growth of neurotrophin-mediated CNS.

Definition

The term that the below defines runs through and is used in this specification, and will be to understanding scope of the present invention and implementing helpful.

" inhibition environment " means the impaired or injured repressed environment of neuronic growth in it as used herein. Suppressing environment is present in the impaired or injured neuronic surrounding environment. Impaired or injured neuron is present in such environment, and it comprises, for example, and with the infringement of CNS or weak relevant disease and the disorder of CNS function. The exemplary state of an illness includes, but not limited to Alzheimer's, Parkinson's, Heng Tingdunshi disease, ALS, apoplexy, traumatic brain injury and spinal cord injury. Alternatively, (being the p75 acceptor combines with molecule in conjunction with the p75 acceptor to suppress environment and be environment in inhibitor complexes of p75 receptors bind in it, for instance, molecule such as MAG or the Nogo-A in myelin source), and this combination causes the inhibition of neurotrophin-mediated neure growth.

Term " treatment effectively " need to mean its individuality once administration, just is enough to produce the active compound of expectation or the amount of pharmaceutical composition. Preferably, clinical significant deficiency can be improved or prevent to the treatment effective dose in the reaction of activity, function and individuality. Alternatively, the treatment effective dose is enough to cause the improvement of the individual clinical great patient's condition. For example, " treatment effectively " mean to be enough to promote to suppress amount or the dosage of the p75 acceptor inhibitor of CNS neure growth in the environment

As used herein, phrase " pharmaceutically acceptable " refers to molecular entity and the composition of " usually being regarded as safety ", for instance, be can tolerate on the physiology and when giving the people, can be typical real estate give birth to irritated or similar inappropriate reaction, such as molecular entity and the composition of gastric disorder causing nausea, the similar symptom such as dizzy. Preferably, term " pharmaceutically acceptable " means to be can be used for animal by management organization's approval of the federation that lists in American Pharmacopeia or other pharmacopeia of usually generally acknowledging or state government as used herein, especially for the people.

Term " carrier " refers to diluent, adjuvant, excipient or the medium of therewith administration of compound. These pharmaceutical carriers can be sterile liquids, and for example water and oil comprise vaseline, animal oil, vegetable oil or synthetic oil of originating, such as peanut oil, soybean oil, mineral oil, sesame oil etc. Water or aqueous salt solution and dextrose hydrate and glycerite preferably are used as carrier, are used in particular for parenteral solution. Suitable pharmaceutical carriers is described in by E.W.Martin among " (Lei Shi pharmacy complete works of " (" Remington ＇ s Pharmaceutical Sciences ").

" individuality " that uses in the literary composition or " patient " be mammal preferably, is more preferably the people, but can be any animal, comprises the laboratory animal under clinical testing or screening or the activity experiment environment. Therefore, as those of ordinary skills hold intelligible, method of the present invention is particularly suitable for any animals administer, particularly mammal, includes but not limited to domestic animal, wild animal and zoologizes.

6.1 TRK receptor modulators: cyclic peptide

The term that uses in the literary composition " cyclic peptide " refers to peptide or its salt, and it comprises: (1) is the interior covalent bond of the molecule between adjacent residues neither; (2) at least one Trk-Receptor recognition sequence RGE (being Arg-Gly-Glu) in the ring ring of described cyclic peptide. Should be appreciated that the preferred peptide of the present invention as Trk receptor stimulating agent or antagonist performance function will suffer restraints, and therefore preferred cyclic peptide. Yet acyclic or " linearity " peptide also are useful (for instance, preparing cyclic peptide of the present invention as intermediate compound). Therefore, the acyclic form that runs through the cyclic peptide that the application describes also is considered to a part of the present invention.

Intramolecular key can be main chain to main chain, side chain to main chain or the side chain key (that is to say that the side chain functionalities of the functional end-group of linear peptides and/or terminal or inner residue can be connected to realize cyclisation) to side chain. Preferred intramolecular key includes but not limited to: disulfide bond, amido link and thioether bond. The several different methods that is used for the cyclisation polypeptide is well known in the art, as can modifying many other that these peptides carry out. Relevant general discussion is referring to International Patent Publication No. WO 01/53331 and WO 98/02452. These ring keies and other modification also can be applied to cyclic peptide of the present invention and derivative compound. For simplicity, cyclic peptide of the present invention usually is illustrated to show specific ring key in this application, and these ring keies may or may not be preferred. Yet other embodiment that comprises these cyclic peptide of other and/or alternative ring key is apparent for those skilled in the art and therefore is considered to a part of the present invention.

In some embodiment, cyclic peptide of the present invention preferably comprises N-acetyl group (that is to say that the amino that is present in this peptide n terminal residue is acetylation, preferably before cyclisation). Alternatively, cyclic peptide of the present invention can comprise N-formoxyl (that is to say, be present in the amino of this peptide n terminal residue by formylated, preferably before cyclisation). Alternatively, the amino that is present in this peptide n terminal residue can be by methylsulfonyl, and is same, preferably before cyclisation. The existence of these end groups can, for example, strengthen some use in cyclic peptide active or stable. In addition, in certain embodiments, cyclic peptide of the present invention can comprise the C-amide groups.

In certain embodiments, the preferred cyclic peptide of the present invention satisfies general formula:

(formula I)

Wherein, Y₁With YX be amino acid residue, its identity is selected independently, and at residue Y₁And Y₂Between have a covalent bond. Component X₁And X₂Choose wantonly, and if present, the combination that they independently are selected from amino acid residue and connect by peptide bond. Therefore, X₁Or X₂Or X₁And X₂, if present, can be the single amino acids residue, perhaps alternatively, be respectively the amino acid sequence that comprises many amino acid residues that connect by peptide bond.

In preferred embodiments, the cyclic peptide that satisfies above-mentioned formula I will be regulated the receptor-mediated activity of one or more Trk-. For example, in certain preferred aspects, the peptide that satisfies formula I will suppress the receptor-mediated activity of one or more Trk-and thereby be the Trk antagonist. In other embodiments, the peptide that satisfies formula I will strengthen the receptor-mediated activity of one or more Trk and thereby be the Trk activator.

Except the RGE consensus sequence, cyclic peptide of the present invention comprises at least one extra residue usually in ring ring, thus X at least among the preferred formula I₁Or X₂Both one of exist. Usually, X₁And/or X₂Size depend on the activity of desired cyclic peptide. For example, when the expectation cyclic peptide is the Trk antagonist, preferred short peptide sequence. Therefore, in these embodiments, X₁And/or X₂Length respectively preferably 0 and about 10 amino acid between, wherein size be about 1,2,3,4 or 5 amino acid residue particularly preferably. In addition, in these embodiments, X₁And/or X₂Length also preferably so select, thereby the magnitude range of cyclic peptide ring is about 5 to about 15 amino acid residues, and more preferably length approximately between 5-10 amino acid residue. Length be about 5-7 amino acid residue size the peptide ring particularly preferably. These extra residues (X among the front formula I namely₁And/or X₂) can be present in one of the N-end of RGE sequence or C-end, perhaps they can be present in the two ends of RGE sequence.

In the preferred cyclic peptide of the present invention, these extra residues derived from the neurotrophin of one or more natural generations (for instance, NGF, BDNF, NT-3, NT-4, NT-5 and NT-4/5) sequence of interior side joint RGE sequence, wherein with or without 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor and/or other modification. Particularly, the existence that derives from the flanking sequence of neurotrophin can help the specific purpose Trk acceptor of cyclic peptide target. Therefore, in the embodiment of the antagonist of expecting acquisition specific T rk acceptor, cyclic peptide of the present invention can comprise side joint in that the N-of RGE sequence is terminal, C-is terminal or the amino acid residue at these two ends, and these amino acid residues are derived from preferentially in conjunction with the flanking sequence of the neurotrophin of target Trk acceptor.

As an example, and also unrestricted, hereinafter Table I has been listed some preferred cyclic peptide, and these cyclic peptide comprise the extra amino acid residue derived from specific neurotrophin, and its identity also is shown in this table. The right hurdle of Table I give neurotrophin preferential in conjunction with (perhaps, or rather, with the highest binding affinity in conjunction with) the Trk acceptor. Therefore, in one embodiment, each cyclic peptide that Table I is listed can both be used for suppressing the Trk acceptor shown in its right hurdle of Table I, next door. Yet, those skilled in the art will appreciate that different neurotrophin parts has some overlapping to the binding specificity of different Trk acceptors. Therefore, the cyclic peptide of listing in the Table I also can be used as the antagonist of other Trk acceptor. As special example, and also unrestricted, the following examples have proved the cyclic peptide N-Ac-that contains the extra residue that derives from neurotrophin NT-4CSRRGEC-NH ₂Be equal to peptide N-Ac-with the extra residue design that derives from respectively neurotrophin NT-3 and NGFCSHRGEC-NH ₂And N-Ac-CFRRGEC-NH ₂Comparing is more effective TrkB antagonist.

Table I: TRK antagonist

The example that is preferably the particularly preferred cyclic peptide sequence of the present invention of Trk antagonist comprises:

CSRRGEC (SEQ ID NO：1)，

N-Ac- CSRRGEC-NH ₂ (SEQ ID NO：2)，

CARRGEC (SEQ ID NO：3)，

N-Ac- CARRGEC-NH ₂ (SEQ ID NO：4)，

CFHRGEC (SEQ ID NO：5)，

N-Ac- CFHRGEC-NH ₂ (SEQ ID NO：6)，

CSHRGEC (SEQ ID NO：7)，

N-Ac- CFHRGE-NH ₂ (SEQ ID NO：8)，

CRGEC(SEQ ID NO:9) and

N-Ac- CRGEC-NH ₂ (SEQ ID NO：10)。

The underscore of each aforementioned aminoacid sequence is partly represented in the peptide by the part of cyclisation." N-Ac " represents acetylizad N-terminal amino group, " NH ₂" expression C-terminal amide base.

In certain embodiments, the relative circlet peptide of the present invention that does not especially preferably contain the important sequence of side joint RGE consensus sequence.These peptides may comprise or may not contain the N-acetyl group and may or may not contain the C-amide groups.The example of the preferred small cyclopeptide of the present invention comprises:

N-Ac- CRGEC-NH ₂ (SEQ ID NO：10)，

N-Ac- KRGED-NH ₂ (SEQ ID NO：11)，

H-C(O)- CRGEC-NH ₂ (SEQ ID NO：12)，

CH ₃-SO ₂-NH- CRGEC-NH ₂ (SEQ ID NO：13)，

N-Ac- CRGEC-Y-NH ₂ (SEQ ID NO：14)，

H-C (O)- CRGEC-Y-NH ₂(SEQ ID NO:15) and

CH ₃-SO ₂-NH- CRGEC-Y-NH ₂(SEQ ID NO：16)。

In other embodiment of the present invention of expectation Trk agonist, preferred usually long peptide sequence.Especially, be at least one " series connection repeats " that the preferred cyclic peptide of the present invention of Trk agonist comprises the RGE motif.Therefore, in satisfying these cyclic peptide of above-mentioned formula I, will there be X ₁And X ₂In at least one, and comprise the 2nd RGE sequence.More specifically, the general formula below these cyclic peptide of the present invention satisfy:

(formula II)

Among the cotype I, Y ₁And Y ₂Be amino acid residue, its identity is selected independently, and at residue Y ₁And Y ₂Between have a covalent bond.Composition Z ₁And Z ₂Choose wantonly, and if present, the combination that they are independently selected from amino acid residue and connect by peptide bond.Composition Z ₀Also choosing wantonly, and if present, is amino acid residue and some combination of connecting by peptide bond thereof.Therefore, Z ₁, Z ₂, Z ₀Or its combination in any, if present, can be respectively one amino acid residue, or alternatively, they can be the sequences that comprises a plurality of amino acid residues that connect by peptide bond.

Except the multiple RGE consensus sequence of connecting, cyclic peptide of the present invention comprises an extra residue usually in its ring ring, so, preferably, Z ₁, Z ₂And/or Z ₀In at least one existence.In the embodiment of the cyclic peptide of expecting the Trk agonist, Z ₁, Z ₂And/or Z ₀Length preferably only about 10 aminoacid respectively, more preferably length only is respectively 1,2,3,4 or 5 amino acid residue.In addition, Z ₁, Z ₂And/or Z ₀Length preferred so select, thereby the length range of cyclic peptide ring is about 8-50 amino acid residue, more preferably approximately 8-25 or about 15-20 amino acid residue.

The cyclic peptide of cotype I is the same, in the cyclic peptide of preferred formula II, and these extra amino acid residues (Z just ₁, Z ₂And/or Z ₀) can be derived from the sequence of neurotrophin (as NGF, BDNF, NT-3, NT-4, NT-5 or NT-4/5) the RGE sequence of one or more natural generations of side joint, described additional amino acid residue has or does not have aminoacid replacement and/or other modification.Especially, the existence that derives from the flanking amino acid residue of specific neurotrophin can help the specific purpose Trk receptor of cyclic peptide targeting.Therefore, in the embodiment of the antagonist of expecting acquisition specific T rk receptor, cyclic peptide of the present invention can comprise the amino acid residue of side joint at one or two connect multiple RGE sequence of N-end and/or C-end, and these side joint sequences can be derived from the neurotrophin of preferential binding purpose target Trk receptor.

As noted above, the present invention's multiple cyclic peptide (comprising the cyclic peptide according to formula II) of preferably connecting has two antiparallel each other RGE sequences that form a line.Therefore, in preferred cyclic peptide according to formula II, composition Z ₀Exist and energy conduct effective " spacerarm part " performance function, thereby these two RGE sequences are formed a line with antiparallel arrangement conformation.In preferred embodiments, Z ₀Length 10 amino acid residues at the most, and preferred length is 5 or amino acid residue still less.Z ₀Preferred size is that about 1,2,3,4 or 5 amino acid residue is long, Z ₀The definite sequence of interior amino acid residue is unimportant.Thereby composition Z ₀May or may not comprise corresponding to amino acid residue sequence from the terminal arbitrary sequence of the N-end of the RGE motif in the natural neurotrophin (as NGF, BDNF, NT-3, NT-4, NT-5 and NT-4/5) or C-.Work as Z ₀When comprising the sequence from neurotrophin really, those sequences may or may not comprise aminoacid replacement and/or modification.

The example that is preferably the particularly preferred cyclic peptide sequence of the present invention of Trk agonist comprises:

CSRRGELAASRRGELC(SEQ ID NO：17)

N-Ac- CSRRGELAASRRGELC-NH ₂(SEQ ID NO：18)

CFHRGEFSIFHRGEFC(SEQ ID NO：30)

CARRGELSARRGELC(SEQ ID NO：31)

CSHRGEYSKSHRGEYC(SEQ ID NO：32)

Cyclic peptide sequence with SEQ ID NOs:30,31 and 32 signs is respectively TrkA, TrkB and TrkC agonist.SEQ ID NO:30 is derived from NGF.SEQ ID NO:31 is derived from BDNF.SEQ ID NO:32 is derived from NT-3.

Cyclic peptide as herein described can comprise L-amino acid residue, D-amino acid residue or its combination in any.As long as guarantee to exist at least one amino and at least one carboxyl in molecule, these aminoacid can be from natural or non-natural source so.Usually preferred α-and beta-amino acids.20 kinds of L-aminoacid that are common in the protein are preferred especially in the present invention.These aminoacid with the abbreviated form sign of their traditional trigrams and single-letter, identify otherwise corresponding D-aminoacid passes through prefix " d " in the text.

In certain embodiments, cyclic peptide of the present invention can comprise with literary composition in the opposite D-amino acid residue sequence of L-amino acid residue sequence that provides.For example, the invention provides and be referred to as riB in the text _AG1And hriB _AG2Some Trk receptor stimulating agent polypeptide of (SEQ ID NOS:40-41), they comprise the sequence of D-aminoacid sequence, and these sequences are to be referred to as B _AGThe reverse sequence of another Trk receptor stimulating agent polypeptide of polypeptide (SEQ ID NO:17).Therefore, except above-described L-amino acid residue polypeptide, the present invention also pays close attention to the polypeptide of the reverse sequence with L-amino acid residue.Therefore, in a preferred embodiment, the sequence of the L-amino acid residue of describing before peptide of the present invention and peptide mimics are included in that comprises Arg-Gly-Glu (i.e. " RGE ") motif.Correspondingly, in an alternate embodiment, the present invention also provides peptide and the peptide mimics that comprises the D-amino acid residue sequence, and it comprises short-term sequence motifs dGlu-Gly-dArg (i.e. " dEGdR ").

Cyclic peptide also can contain the derivant of one or more not common amino acids (for example 4-hydroxyproline or oxylysine), organic acid or amide and/or common amino acid; for example have C-terminal carboxylic esterification (as benzyl esters, methyl ester or ethyl ester) or amidated aminoacid and/or have the aminoacid of N-terminal amino group (as acetylation or carbalkoxylation effect); having or do not have multiple side chain modifies and/or replaces (be that methylation, benzyl turn usefulness, the effect of t-butylation, tosyl into and turn usefulness, carbalkoxylation effect into, or the like).Preferred derivant comprises and has the N-acetyl group aminoacid of (amino of linear peptides N-end is acetylation before the cyclisation thereby represent) and/or C-terminal amide base (carboxyl terminal of the linear peptides that cyclisation just is preceding is by amidatioon).May be present in the residue that is different from common amino acid in the cyclic peptide includes but not limited to: penicillamine, β, β-tetramethylene cysteine, β, β-pentylidene cysteine, β-Qiu Jibingsuan, β, β-pentylidene-β-Qiu Jibingsuan, 2-sulfydryl benzene, 2-mercaptoaniline, 2-sulfydryl proline, ornithine, DAB, alpha-Aminoadipic acid, m-amino methyl benzoic acid and α, β-diaminopropionic acid.

Cyclic peptide as herein described can synthesize by means commonly known in the art, comprises recombinant DNA method and chemical synthesis.Chemical synthesis can adopt the liquid phase of standard or solid-phase peptide synthetic technology to carry out usually, and wherein the peptide bond bonding is by an amino acid whose alpha-amido and the direct condensation of another amino acid whose α-carboxyl, and follows the removal of hydrone and take place.By the synthetic reactivity that needs to suppress first amino acid whose amino and second amino acid whose carboxyl of the peptide bond of above-mentioned direct condensation.This substituent group of sheltering must allow their easy removals, and does not induce the decomposition of unsettled peptide molecule.

In liquid phase is synthetic, can use multiple coupling method and blocking group (referring to Gross ﹠amp; Meienhofer edits, " peptide: analyze, synthetic, biology " (" The Peptides:Analysis, Synthesis, Biology ") 1-4 rolls up (Academic Press, 1979); Bodansky ﹠amp; Bodansky, " peptide is synthetic to be put into practice " (" The Practice of Peptide Synthesis ") second edition (Springer Verlag, 1994)).In addition, might carry out intermediate purification and linear amplification.Those of ordinary skills will appreciate that synthetic consideration main chain and side chain protected group and the activation method of needing of liquid phase.In addition, during fragment condensation, carefully selecting fragment is essential to racemization is minimized.Particularly, when the residue of coupling, can be observed the racemization of high percentage ratio as Phe-Gly.Yet this situation is uncommon.The consideration of dissolubility also is a factor.

The solid phase method of peptide synthesis uses insoluble polymer as holder during organic synthesis.The peptide chain of polymer support allows to use simple washing and filtration step to replace the purification of requiring great effort in the intermediate steps.The solid phase method of peptide synthesis usually can be according to people such as Merrifield, " Journal of the American Chemical Society " (J.Am.Chemin.Soc.) 1963, and the method for 85:2149 is carried out.These methods comprise uses shielded aminoacid to assemble linear peptide chain on the resin holder.Solid phase method of peptide synthesis typical case uses Boc or Fmoc strategy.The Boc strategy uses 1% cross-linked polystyrene resin.Standard blocking group to amino functional is tertbutyloxycarbonyl (Boc) group.This group can be removed with for example strong acid diluent of 25% trifluoroacetic acid (TFA).Next Boc-aminoacid is typically utilized dicyclohexylcarbodiimide (DCC) to be coupled on the glycyl resin.After assembling is finished, handle peptide-resin with anhydrous HF and connect and the release free peptide to cut benzyl esters.Usually by the blocking groups sealing derived from benzyl, these blocking groupses are also decomposed by HF side chain functionalities between synthesis stage.Use suitable solvent that free peptide is extracted from resin subsequently, and carry out purification and sign.New synthetic Toplink is purified by example gel filtration, HPLC, Partition Chromatography and/or ion-exchange chromatography, also can be characterized by general analysis of for example matter or amino acid sequence analysis.In the Boc strategy, the peptide of C-terminal amideization can use benzhydryl amine or methyldiphenyl methyl amine resin to obtain, and they directly produce peptide amide after HF decomposes.

In the operation of Tao Luning, the selectivity that the side chain blocking groups is connected with peptide resin depends on the difference of acidolysis resolution ratio in the above.The Orthoganol system is introduced into, and wherein be connected for the reagent that is used to remove blocking group in each synthesis step with peptide resin be very stable to the side chain protected group.Modal 9-fluorenylmethyloxycarbonyl (Fmoc) approach that relates to of these methods.In the method, the side chain protected group is connected for the secondary amine that is used for decomposing N-α-Fmoc group with peptide resin be very stable.Side chain protected is connected with peptide resin by gentle acid hydrolysis and decomposes.Make the Merrifield resin be not suitable for the Fmoc chemistry with contacting repeatedly of base, and use the p-alcoxyl benzyl esters that is connected on the resin usually.Usually use TFA to finish deprotection and decomposition.

It will be appreciated by those of ordinary skill in the art that and in solid-phase synthesis, run through entire synthesis process that deprotection and coupling reaction must be carried out thoroughly, and the side chain blocking groups must be stable.In addition, when the small-scale production peptide, solid-phase synthesis is normally only.

The terminated acetylated interaction energy of N-was finished by final peptide and acetic anhydride are reacted before decomposing from resin.Carbon-amidation can use appropriate resin for example methyldiphenyl methylamine resin use the Boc technology and finish.

After linear peptides is synthetic, follow or do not follow N-acetylation and/or C-amidation, can realize cyclisation by multiple technologies well known in the art.In one embodiment, key can produce between the amino acid side chain that reacts.For example, set out, come this Toplink of oxidation to form disulphide bridges by using any in the several different methods to comprise two linear peptides that contain the mercaptan residue.In a kind of the method, use alkalescence or neutral water-bearing media, can produce disulfide bond through the air oxidation of time mercaptan in a few days.This peptide uses with high dilution, thereby makes gathering and intermolecular side reaction drop to minimum.The shortcoming of this method is slow, but advantage is only to produce by-product H ₂O.Alternatively, can use for example I of strong oxidizer ₂And K ₃Fe (CN) ₆Form disulfide bond.Thereby those of ordinary skills will appreciate that reaction and must be carried out carefully making that the responsive side chain of Met, Tyr, Trp or His is not oxidized.The cyclic peptide that produces by this method need use standard techniques to come purification, but this Oxidation is applicable to acid pH.

Thereby oxidant also allows deprotection/Oxidation coexistence of the linear precursor of suitable S-protection to avoid that free cysteine is too early, nonspecific Oxidation.

Unlike I ₂And K ₃Fe (CN) ₆, DMSO is a kind of oxidant of gentleness, it does not make above-mentioned nucleophilic aminoacid produce the oxidation side reaction.DMSO and H ₂O easily mixes under all concentration, and Oxidation can carry out to neutral pH in acidity, is accompanied by harmless by-product.Alternatively, can use methyl trichlorosilane-diphenyl sulfoxide,, and not influence other nucleophilic aminoacid with the deprotection/Oxidation of the coexistence of the S-Acm, the S-Tacm that carry out cysteine or S-t-Bu as oxidant.The formation of intermolecular disulfide bond does not produce polymerizate.

The suitable mercaptan residue that contains that is used for these method for oxidation includes but not limited to: cysteine, β, Beta-Dimethylcysteine (penicillamine or Pen), β, β-tetramethylene cysteine (Tmc), β, β-pentylidene cysteine (Pmc), β-Qiu Jibingsuan (Mpr), β, β-pentylidene-β-Qiu Jibingsuan (Pmp), 2-sulfydryl benzene, 2-mercaptoaniline and 2-sulfydryl proline.

Show to those skilled in the art and suggestion in each of the representational structural formula of these that list, can adopt any one above-mentioned residue that contains mercaptan in front, to replace in the described residue that contains mercaptan one or two.

In further embodiment, cyclisation can obtain by the formation of amido link.For example, peptide bond forms between functional group's (being the amino and the carboxyl terminal of linear peptides before the cyclisation) endways.The example of these peptides comprise c ( SRRGE) (SEQ ID NO:33), c ( ARRGE) (SEQ ID NO:34), c ( FHRGE) (SEQ ID NO:35) and c ( SHRGE) (SEQ ID NO:36).Particularly preferred example with peptide of this cyclic amides key be peptide c ( SRRGELSRRGEL) (SEQ IDNO:39).This peptide is described in the following examples, and it is known as hB in the text _AG2Peptide.In another such embodiment, linear peptides comprises D-aminoacid.For example, the following examples have been described another preferred peptide, are referred to as to do hriB _AG2Peptide.This peptide contains cyclic amides key recited above, and it comprises following D amino acid residue sequence: c[dLdEdGdRdRdSdLdEdGdRdRdS] (SEQ ID NO:40).Alternatively, cyclisation can be by connecting an end and residue side chain or use two side chains to finish, just as KRGEDLike that, have or do not have terminal acetyl group of N-and/or C-terminal amide among (SEQ ID NO:37) or the KSRRGED (SEQ IDNO:38).The residue that can form lactam bond comprises lysine, ornithine (Orn), alpha-Aminoadipic acid, m-amino methyl benzoic acid, α, β-diaminopropionic acid, glutamic acid or aspartic acid.

The method that forms amido link is well known in the art, and based on the chemical reactivity principle of good establishment.In such method, the lactams of carbodiimides mediation forms and can finish by the reaction of carboxylic acid and DCC, DIC, EDAC or DCCI, thereby causes forming the O-acyl group urine that can finish cyclisation immediately with the free amino group reaction.The forming of the inactivation N-acyl group urine that is produced by O → N migration can convert O-acyl group urine to active ester by the reaction with N-hydroxy compounds such as I-hydroxybenzotriazole, 1-N-Hydroxysuccinimide, 1-hydroxyl norborene Methanamide (1-hydroxy norbornene carboxamide) or ethyl 2-oximido-2-cyano-acetate and prevent.Except O → N migration was minimized, these additives also served as catalyst and participate in reducing racemic effect during cyclisation.Alternatively, cyclisation also can use the azide method to carry out, and reactive in the method azide intermediate generates from Arrcostab by hydrazides.The hydrazides effect of terminal ester need use the t-butyl to protect side chain carboxyl group function in the acidylate composition.This limitation performance is overcome by using diphenyl phosphinylidyne acid (DPPA), and this diphenyl phosphinylidyne acid is through directly providing azide with carboxyl reaction.The long response time of azide and limited the effectiveness of this method by their formation of isocyanates of dismutation reaction.The mixed anhydride method that lactams forms is widely used owing to removing byproduct of reaction easily.Anhydride is through carboxylate anion and methyl chloride formic acid esters or pivaloyl chlorination thing reaction formation.The attack of amino group is directed to the carbonyl carbon of acidylate component subsequently by the spatial volume of sub-effect of the power supply of alkoxyl or the t-butyl by trimethyl-aceyl chloride, this has blocked the attack on wrong carbonyl.The mixed acid anhydride that has phosphoric acid derivatives is also successfully used.Alternatively, cyclisation can use Acibenzolar to finish.The existence of electron-withdrawing substituent has improved their sensitivity to ammonolysis on the ester alkoxyl carbon.The height reactivity of the ester of paranitrophenol, N-hydroxy compounds and many halogenations phenol makes these " active ester " can be used for the synthetic of amido link.Several years of past developed benzotriazole base oxygen base three-(dimethylamino) phosphorus hexafluorophosphoric acids (benzotriazolyloxytris-(dimethylamino) phosphonium hexafluorophosphonate) (BOP) and congeners as useful coupling agent.Their performance is better than the performance that the good carbodiimides amido link of establishing forms reaction usually.

In further embodiment, between side chain that contains the mercaptan residue and suitable deutero-a-amino acid, can form thioether bond.For example, lysine side-chain can pass through the carbodiimides coupling method (DCC EDAC) is coupled on the bromoacetic acid, and subsequently with above-mentioned arbitrary mercaptan residue formation thioether bond that reacts that contains.In order to form thioether bond, any two side chains that contain mercaptan can both react in DMF with Bromofume and diisopropylamine.The example that contains the mercaptan key comprises:

With

Wherein X can be (CH ₂) ₄, CH ₂Or

Cyclisation also can be used δ ₁δ ₁-.delta.1.delta.1'-Ditryptophan. (being Ac-Trp-Gly-Gly-Trp-OMe) is realized, and is as follows:

Structure of being quoted among the application and formula only are used for illustration purpose, do not constitute the restriction to cyclic peptide described in the application.

6.2 TRK-receptor pharmacophore

Be the designed peptide analogies, the three dimensional structure that obtains the pharmacophore of one or more above-mentioned cyclic peptide is useful.Term " pharmacophore " refers on the chemical compound to be arranged in three-dimensionally with the complementary mode of target proteins, and is responsible for because of the set of chemical compound in conjunction with the bioactive functional group that target proteins produced.Useful three-dimensional Pharmacophore Model is best derived from the crystallization or the nuclear magnetic resonance, NMR structure of target, but also can based on the structure of relevant target or derive from previous discovery serial reactive compound 3D-QSAR and derived from homology model.

The invention provides the pharmacophore (being the three-dimensional conformation of neurotrophin consensus sequence RGE in these peptides) of some representative cyclic peptide.These three dimensional structures provide and have instructed design most effectively and optimize the required information of peptide mimics.

In one embodiment, the three dimensional structure of cyclic peptide uses the X-radiocrystallgraphy to measure usually.These technology are known and in the routine techniques scope of this area.For example, referring to, Cantor ﹠amp; Schimmel, " biophysical chemistry " (Biophysical Chemistry) 1980 I-III volume) W.H.Freeman and Company (particularly 1-13 chapter in the I volume and the 13rd chapter in the II volume).Also referring to, " polymer crystallization " (Macromolecular Crystallography), PartsA-B (Carter ﹠amp; Sweet edits) In: " Enzymology method " (Methods Enzymol.) 1997, the 276-277 volume; Jan Drenth, " protein X-ray crystallography principle " (Principles ofProtein X-Ray Crystallography) (New York:Springer-Verlag, 1994).

Term " crystal " is commonly referred to as molecule any orderly (or partial order) at least three-dimensional arrangement.Preferably, the ordering of molecule at least enough produces sensitive x-ray diffraction pattern in the crystal, thereby can measure the three dimensional structure of this molecule.

Intracrystalline molecule can be any form, should be appreciated that crystal can comprise the molecule that has only a kind of form, also can comprise multiple multi-form molecule.In preferred embodiments, crystal of the present invention comprises at least a biomolecule, for example the cyclic peptide of describing in 6.1 joints in front.Crystal of the present invention even can comprise the complex or the set of two or more albumen or other biomolecule.For example, crystal can comprise ligand molecular such as neurotrophin, and it is combined on acceptor molecule such as the Trk receptor.Typically, the crystal that comprises all glairy biomolecule also will comprise other molecule, such as the molecule of solvent (hydrone for instance) and/or salt.Other molecule for example medicine, drug candidates or protein-bonded chemical compound also can be present in the crystal.

In fact, the crystal structure with the binding structural domain of the Trk receptor of neurotrophin complexation exists in the prior art already.Referring to, for example, people such as Wiesmann, " nature " (Nature) 1999,401:184; With people such as Banfield, Structure (Camb.) 2001,9:1191.The coordinate of these X-ray structures is easy to obtain, for example, from＜www.rcsb.orb〉Protein Data Bank of (accession number is respectively: 1www and 1hcf) obtains.Therefore, in particularly preferred embodiments, set forth among this embodiment hereinafter, use the crystal X-ray structure of the neurotrophin of the suitable Trk receptor (or its fragment) of combination to determine pharmacophore structure of the present invention.These three dimensional structures can be used to design peptide mimics of the present invention subsequently, or alternatively, are used to design other cyclic peptide that is likely the Trk regulator.

Alternatively, the three dimensional structure of cyclic peptide can use nuclear magnetic resonance, NMR well known in the art (NMR) technical measurement usually.The NMR data are obtained preferably and are carried out in the aqueous system of simulating physiological condition closely, thereby guarantee to obtain dependency structure.In brief, the utilization of NMR technology have magnetic moment or a magnetic spin some atomic nucleus (for example, ¹H, ¹³C, ¹⁵N and ³¹P) magnetic is surveyed the chemical environment of this nuclear.The NMR data can be used to measure the distance between the intramolecularly atom, and this can be used for deriving threedimensional model or molecule.

For measuring the three dimensional structure of cyclic peptide (and candidate's peptide mimics of following discussion), preferably use proton N MR.More specifically, when molecule was placed high-intensity magnetic field, the hydrogen atom of two spin states was no longer decayed.The spin that is arranged in parallel with magnetic field will have lower energy, and will have higher energy with the spin of magnetic field arranged anti-parallel.During balance, the spin of hydrogen atom will distribute according to Boltzmann and decide the increase of rate equation.This balance of spin total amount can be perturbed to excited state by the pulse of employing wireless electricity frequency (RF).When nuclear returned back to poised state, they sent the measured RF radiation of energy.To depend on the branch subenvironment of this nuclear from each radiating definite frequency of authorizing out, and (except those have the atom of same molecular environment) all is different concerning each atom.These different frequencies obtain with respect to contrast signal, and are known as chemical shift.The character of applied RF pulse, persistent period and combination can have very big variation, and by selecting suitable pulse combined, those of ordinary skills can detect the character of different molecular.

Measure for three dimensional structure, one-dimensional NMR spectrum is normally not enough, because may obtain the limited information relevant with conformation.One-dimensional NMR is normally used for proving intramolecular connectedness, and generates the fragmentary data of the inboard chain orientation of related peptides.Two dimension NMR spectrum is more useful in this respect, and allows the fuzzy conformation of measuring side chain to side chain interaction and peptide main chain.

Two dimension NMR spectrum is rendered as profile diagram usually, and wherein, diagonal is to be produced by the interaction between the coupled hydrogen atom of direct invariant corresponding to one-dimensional NMR spectrum away from cornerwise intersection peak.Two dimension test generally includes warming up period, period of expansion (wherein being " marked " when they advance with the XY plane according to its chemical shift from being spun on), mixing period (during taken place with other spin relevant) and detection period (during report free induction decay).

Two dimension NMR method is famous with the dependency relation of surveying during mixing period.DQF-COSY (the filtering correlation spectrum of two quantum (double quantum filtered correlation spectroscopy)) analyzes the peak value that has provided between the hydrogen atom that connects by one or two other atom covalence.The relevant spectrum of Ovshinsky nuclear effect (Nuclear Overhauser Effect Spectroscopy) (NOESY) has provided that the space is close together, even by a large amount of peak values that insert between the paired hydrogen atom that atoms connect.Total correlation spectrum (total correlation spectroscopy) (TOCSY) in, observed the dependency relation between all protons of shared coupling gametophyte, no matter and their whether directly couplings each other.The relevant spectrum of rotating coordinate system Ovshinsky (Rotating-frame Overhauser Spectroscopy) (ROESY) is tested the rotating coordinate system analog that can be considered to NOESY, and produces peak value between the paired hydrogen atom that is close together in the space.Method that can one or more are such and necessary water peak pressure system (water-suppression) technology such as WATERGATE (water valve control) and water revolution (flip-back) are used in combination, thus under aqueous conditions the three dimensional structure of mensuration cyclic peptide or candidate's peptide mimics.These technology are known, and the resonance to inhibition solvent (HDO) is essential during obtaining the NMR data.

For instance, TOCSY and NOESY all can be applicable to representational cyclic peptide and measure conformation and distribution.Can suppress aqueous solvent resonance by using the WATERGATE program.The water rotary pulse also can be used in the latter stage of the mixing period of TOCSY and NOESY test, thereby the water signal is maintained poised state, and the loss of amide proton resonance is dropped to minimum, wherein the loss of amide proton resonance be since during the approaching neutral Ph condition used in test (Ph 6.8) their quick exchange cause.Can use and use the spectroscope software of square cosine window function in two directions to process the NMR data.Baseline correction can be applied to use NOESY, ROESY and the TOCSY spectrum of standard Bruker multinomial method.

In the incorporation time several times of change from 80ms to 250ms, can obtain the NOESY data.Can obtain the NOESY of shorter incorporation time, thereby guarantee in the NOESY spectrum that longer incorporation time is obtained, not have spreading effect.Distance between proton can be measured from 250ms NOESY usually.The sequence-specific of proton resonance distribute two results that can utilize TOCSY and NOESY data by standard method (referring to Wuthrich, " protein and nucleic acid nuclear magnetic resonance, NMR " (NMR of Proteins andNucleic Acids), Wiley ﹠amp; Sons, New York, 1986) measure.

For the calculating of conformation, the unified distance that is limited to the 1.8-5.0 dust up and down that NOE intersection peak value can be converted at first and NOE intensity is irrelevant.NOE is apart from being improved repeatedly by the NOEs with test that relatively calculates in different incorporation times.Although preferred use initial based on having of NOE of the very loose upper limit (5 dusts for instance) thus distance allow and produce and the corresponding to more complete conformation of test data, but this improvement is most to be center (people such as Wang with the PEPFLEX-II program, " protein chemistry technology " (Techniques in Protein Chemistry) IV, 1993, the 569th page of Evaluation of NMR Based Structure Determination for Flexible Peptides:Application to Desmopressin).Dihedral angle constraint can by ³The value of JC α H coupling constant is derived.It is intrafascicular approximately to explain the conformation elasticity of peptide that the deviate of 40 degree can be added to each dihedral angle.Geometric distance calculates and can use the ECEPP/2 data base (the fixedly bond distance and the bond angle that provide in 31:11551-11557) carry out for people such as Ni, " biochemistry " (Biochemistry) 1992.ω-angle is fixed on 180 degree usually, but all other dihedral angles are variable during structure optimization.

Use Monte Carlo method (the Ripoll ﹠amp of constraint distance; Ni, " biopolymer " (Biopolymers) 1992,32:359-365; Ni, " nuclear magnetic resonance, NMR magazine " be B 1995 (J.Magn.Renon.), 106:147-155) can make the structure that has minimum constraint interference (lowest eonstraint violations) stand energy minimization, thereby and improve and include the ECEPP/3 field of force (people such as Ni in, " molecular biology magazine " (J.Mol.Biol.) 1995,252:656-671).During the constraint Meng Kateluo of ECEPP/3 energy minimized, all ionizing groups were processed into charged.Electrostatic interaction energy between all electric charges shields by using the electrolyte that relies on distance, thus the shortage of explanation solvent effect in the conformation energy calculates.In addition, the interaction energy of hydrogen bonded is dropped to 25% of total scale, yet Van der Waals force and static project but remain on full width.These special handlings help to guarantee that conformation is sought mainly by experimental NMR constraint instructs, and guarantee the conformation of calculating not too be partial to by rule of thumb the conformation energy parameter (people such as Warder, " FEBS communication " (FEBS Lett.) 1997,411:19-26).

The low-yield peptide conformation that derives from the Monte Carlo statistic algorithm can be used to the NOE simulation with the proton of identifying the nearest visual NOEs of not tool and series guarantee to calibrate again apart from the upper limit.Comprise that deriving from the series apart from lower limit that lacks NOE improves next circulation that the NOE distance is used to the Monte Carlo statistic algorithm, until obtaining producing the spectrographic final conformation of simulation NOE near the experiment that is observed (people such as Ning, " biopolymer " (Biopolymers) 1994,34:1125-1137; People such as Ni, " molecular biology magazine " (J.Mol.Biol.) 1995,252:656-671).Theoretical NOE spectrum can utilize the 1.5ns based on peptide molecular weight and experimental temperature to roll and calculate (Cantor ﹠amp correlation time; Schimmel (1980) " biophysical chemistry " (Biophysical Chemistry), W.H.Freeman﹠amp; Co., San Francisco).All candidate's peptide conformations are included tension and relaxation matrix-analysis method (the Ni ﹠amp of the population mean of change conformation in identical weighting; Zhu, " nuclear magnetic resonance, NMR magazine " be B1994 (J.Magn.Renon.), 102:180-184).But NOE simulation also incorporating parametric is moved with the part of explanation methyl and proton dwindles the effect of the not exclusively lax decay of (demagnitizations) (people such as Ning, Biopolymers 1994,34:1125-1137).The NOE intensity that is calculated with all chemical shifts of having differentiated proton resonance distribute, estimation live width and coupling constant be scaled two-dimentional FID ＇ s (Ni, " nuclear magnetic resonance, NMR magazine " be B 1995 (Magn.Renon.), 106:147-155).The FIDs that calculates can be scaled mimic NOESY spectrum with the used same treatment program of experiment NOE DS.

6.3 TRK receptor modulators: peptide mimics

As noted above, peptide mimics is the adorned chemical compound of RGE sequence at least a portion cyclic peptide in it, and is substantially the same with the three dimensional structure of RGE sequence thereby the three dimensional structure of this peptide mimics keeps.Peptide mimics can be a peptide analogues, and these peptide analogues itself are the cyclic peptide that contains one or more replacements or other modification in the RGE sequence.Alternatively, the part of RGE sequence can be replaced with non-peptide structure at least, thereby the three dimensional structure of this cyclic peptide is kept basically.In other words, in the RGE sequence, two or three amino acid residues can be replaced with non-peptide structure.In addition, other peptide moiety of cyclic peptide can, but unessential, replace with non-peptide structure.Peptide mimics (peptide and non-peptide analogues) can have improvement character (as, the proteolysis of reduction, the bioavailability of enhanced retentivity or raising).Peptide mimics has improved oral availability usually, and this makes them be particularly suitable for treating for example disease of cancer.Should be noted that peptide mimics may or may not have similar two-dimentional chemical constitution, but total common Three Dimensions Structure and geometry.Each peptide mimics can further have the extra binding member of one or more uniquenesses.The invention provides the method that is used to differentiate peptide mimics.The multiple modification that peptide is modified (modification of the cyclic peptide of describing before being included in) is well known in the art, and can be used to produce the peptide mimics chemical compound.Referring to, for example, international patent WO01/53331.These modifications also can be used for the present invention and produce the peptide mimics chemical compound, and following specific modification.

All peptide mimicses that the application provides have the three dimensional structure similar basically to the three dimensional structure of above-mentioned cyclic peptide.Generally speaking, if the root average variance (RMSD) of the atomic coordinates of two three dimensional structure pharmacophore is less than or equal to 1 dust, as use QUANTA program (QUANTA, be attained at Molecular Simulations Inc., San Diego, Calif.) Nei molecular mimicry module calculated like that, so, it is similar basically on the structure that these two three dimensional structures are considered to each other.All peptide mimicses provided herein have at least one low-yield three dimensional structure similar in fact at least one low-yield three dimensional structure of above-mentioned cyclic peptide.

Low energy conformations can be by using, and for example, (conformational energy 4:187-217) calculates to be discerned the CHARMM program for people such as Brooks, " chemistry magazine " (J.Comput.Chem.) 1983.The energy spectral term comprises bonding and nonbonding spectral term, and these spectral terms comprise bond distance's energy, angle energy, dihedral angle energy, Van der Waals energy and electrostatic energy.Obviously, conformational energy also can use any multiple other commercially available quantum mechanics or molecular mechanics program to calculate.The conformational energy of low-energy configuration is within the global minimum of 52kcal/mol.

The low energy conformations of candidate's peptide mimics is compared with the low energy conformations of cyclic peptide (for example, as measuring), determine that how approaching the conformation of candidate's analogies and the conformation of cyclic peptide have by NMR or X-radiocrystallgraphy.These relatively in, should be specifically noted that location and orientation corresponding to the element of crucial side chain.If at least one material standed for low energy conformations similar basically to the comformation in solution of cyclic peptide (just differing from 1 dust or root average variance (RMSD) still less), this candidate compound is considered to peptide mimics so.In these are analyzed, the low energy conformations of candidate's peptide mimics can in the solution, for example by the TIP3P water model of presenting hydrone (people such as Jorgensen, " chemical physics magazine " (J.ChemPhys.) 1983,79:926-935) use CHARMM molecular mechanics and molecular dynamics program (people such as Brooks, " chemistry magazine " (J.Comput.Chem.) 1983 4:187-217) studied.The CHARM22 field of force can be used for presenting the peptide mimics of design.

For instance, can use the binding energy of two programs to differentiate low energy conformations.First program comprises the dynamics simulation method of mimic annealing molecule.In this program, system's (it comprises the peptide mimics and the hydrone of design) is heated to more than the room temperature, 600K preferably approximately, and simulate 100 picoseconds (ps) or longer time; Reduce to 500K then gradually, and simulation 100ps or longer time; Reduce to 400K then gradually and simulate 100ps or the longer time; Reduce to 300K gradually and simulate 500ps or the longer time.Track record is used for analyzing.This mimic annealing operation has effective conformation search capability as everyone knows because of it.

Second program comprises uses self molecular dynamics (SGMD) method (Wu; Wang, " physical chemistry magazine " (J.Physical Chemistry) 1998,102:7238-7250).The SGMD method has been proved to be has extremely enhanced conformation search capability.Use the SGMD method, simulation can be carried out 1000ps or longer time at 300K, and track record is used for analyzing.

Can use QUANNTA molecule modeling bag to carry out conformational analysis.At first, can use the track that produces from the molecule dynamic simulation to carry out cluster analysis.Can select the lowest energy conformation as representational conformation from each cluster, and the cluster of itself and other conformation can be compared for this cluster.Through cluster analysis, can discern main conformation cluster, and the comformation in solution of itself and cyclic peptide is compared.The conformation comparison can use the molecular mimicry module in the QUANTA program to carry out.

Similarity in the structure also can pass through the visual comparison with the three dimensional structure of graphic form demonstration, or relatively estimates by arbitrary multiple calculating.For example, can determine the atom equivalent in the three dimensional structure of peptide mimics and cyclic peptide, and use the match operation to set up similarity level.Employed in the literary composition " atom equivalent " is the atom of one group of conservation in two structures." match operation " can be any process, and by this process, thereby the structure of candidate compound is by the best fit of translation and rotation realization and cyclic peptide structures.The match operation can be strict match operation (for instance, the three dimensional structure of cyclic peptide is maintained fixed, thereby and the three dimensional structure of peptide mimics can and rotate the best fit of realizing with cyclic peptide by translation).Alternatively, match operation can be used the least square fitting algorithm, and the best translation and the rotation of the compound structure that is applied to move waited in its calculating, thereby the root average variance of the match of the reciprocity homoatomic of specified one-tenth is a minima.Preferably, the user can establish the atom equivalent, and the match operation can use arbitrary multiple obtainable application software (for instance, deriving from Molecular Simulations Inc., San Diego, the QUANTA of Calif.) to carry out.The three dimensional structure that is used to set up the candidate compound of basic similarity can (for instance, use NMR technology described herein or X-radiocrystallgraphy) by experiment to be determined, also can use, and for example, method computer provided herein generates.

Can design some peptide mimics based on cyclic peptide structures.For example, these peptide mimicses can be simulated at cleavable amido link (amido link isostere) local topology figure on every side.The example of backbone modifications provides in Fig. 3 A and 3B (also referring to, Fig. 4 A-4B among the WO 01/53331).These analogies usually atom pair mate the peptide main chain atomically, have kept simultaneously with binding site to produce the important function that contacts.The amido link analogies also can comprise mixing of non-common amino acid or dipeptides succedaneum.The example of these non-common amino acids and dipeptides succedaneum is set forth in Fig. 4 (also seeing the Fig. 5 among the WO 01/53331) at this.Also have other example be well known in the art (referring to, for example, people such as Gillespie, " biopolymer " (Biopolymers) 1997 is among the 43:191-217).With conformation more flexibly peptide bond compare, think that the inflexible structural detail of conformation that sees in this analoglike thing causes its entropy driving force combination with the height facilitation.With respect to parent's peptide, backbone modifications also can be given the cracked metabolic stability of peptidase.Other peptide mimics can be the secondary structure analogies.These peptide mimicses adopt non-peptide structure to replace concrete secondary structure usually, for example β-corner, beta sheet and α-anglec of rotation (referring to Fig. 5).

Be the designed peptide analogies, can use the heuristic rule that grows up by experience to come system to modify cyclic peptide.In this modification, run through repeatedly development usually and collect different types of empirical data.As above mention especially, the optimal efficacy in the peptide mimics design needs the three dimensional structure of pharmacophore.

The pharmacophore that the application provides allows the peptide mimics design based on structure, for example, modifies by above-mentioned peptide support.Some peptide mimics can be discerned by the vision-based detection that one or more pharmacophore are compared with neurotrophin RGE conformation.Use the knowledge of the structure-activity relation of cyclic peptide, also can come the designed peptide analogies based on the visual comparison of the three dimensional structure of cyclic peptide pharmacophore and candidate compound.Structure is imitated to study and has been established important binding member in the cyclic peptide, and makes the possibility that develops into of Pharmacophore Model.She Ji peptide mimics should keep these binding members in this way.

Also can be according to (--S--CH2--C (O)--) replaces S--S--) designed peptide analogies of disulfide bond (--with thioether bond.Disulfide bond is not very stable usually, because it is reduced under acid condition easily.Partly replace the stability that disulfide bond can significantly improve peptide with thioether, and therefore improve oral availability.

As substituting of designing by vision-based detection, can use combinatorial chemistry technique to set up library (for instance, containing hydantoin and/or oxygen diethylenediamine compound).Combinatorial chemistry technique makes by adding the parallel possibility that becomes that clear and definite chemical constituent is carried out organic compound with highly reliable chemical reaction and automatic device system ground.A large amount of library of compounds be the institute that can finish in a site might react and can finish in the site of second, third or greater number the result of the combination that might react.Combinatorial chemical method can be to produce hundreds and thousands of ten thousand new chemical compounds attached to the mixture on the carrier or in the mode of individualized compound potentially.

Pharmacophore can be used to simplify these chemical library screening.For example, the library is synthetic can be concentrated on the library member who has with the interactional maximum likelihood of target, rather than produce the used of each library may member's (producing the chemical compound of difficult quantity).Integrated application can make collaborative raising of efficient of drug development based on structure Design and combinatorial chemistry technique.

Other peptide mimics seemingly with original peptide unrelated compounds, but they contain the functional group that is positioned on the non-peptide support that serves as topological analogies.This class peptide mimics is referred to as " non-acyltransferase polypeptide analog " in the text.These peptide mimicses can use the library screening of a large amount of chemlines to be identified.These screenings use the three-dimensional conformation of pharmacophore to retrieve these data bases in three dimensions.In this retrieval, can use the single 3 D structure as Pharmacophore Model.Alternatively, Pharmacophore Model can produce by considering the crucial chemical structure characteristic that is present in a plurality of three dimensional structures inside.

Can use arbitrary multiple three-dimensional structure database to carry out these retrievals.By producing the three dimensional structure of compound database, and store three dimensional structure, can prepare three-dimensional structure database with the form of machine-readable data coded data storage medium.Three dimensional structure can demonstrated 3-D graphic and install on the machine of the programmed instruction that uses these data and demonstrated.In preferred embodiments, three dimensional structure provides with the form of the coordinate system that defines this three dimensional structure.

Preferably, the 3D-data base comprises at least 100,000 chemical compound, wherein have relatively simple chemical constitution little, non-acyltransferase polypeptide molecule is preferred especially.The 3D coordinate of chemical compound also is very important with describing rightly accurately among the data base.The 3D-data base of National Cancer Institute (NCI) (people such as Milne, " chemical information computational science magazine " (J.Chem.Inf.Comput.Sci.) 1994,34:1219-1224) and obtainable chemical guide (ACD; Available from the MDL information system, San Leandro Calif.) is two very outstanding data bases, and they can use molecule modeling discussed above to produce three-dimensional structure database.For flexible molecule, they may have several low energy conformations, and a plurality of conformations of storage and retrieval therefore are supposed to.Chem-X program (Oxford Molecular Group PLC; Oxford UK) can retrieve the conformation of thousands of even millions of flexibility compounds.This ability of Chem-X provides tangible superiority when handling the chemical compound that can take a plurality of conformations.Add up to 465,000 chemical compound although the NCI-3D data base comprises at present, make in this way, can in 3D-pharmacophore retrieving, retrieve billions of conformations.

Pharmacophore retrieval typical case comprises three steps.First step is to produce the pharmacophore inquiry.These inquiries are formed by the evaluation to the critical distance in the cyclic peptide three dimensional structure.The inquiry of application target pharmacophore can carry out the choosing of range word knotter screen in the data base, thereby the chemical compound of required geometrical constraint is satisfied in identification.In other words, identified the chemical compound that satisfies specific critical paired distance.After chemical compound selected step by the range word knotter screen, next this program checked whether this chemical compound satisfies the substructure requirement of regulation in the pharmacophore inquiry.After chemical compound is checked by this substructure, carry out conformational analysis at last.In this step, produce conformation and require to estimate with regard to how much of regulation in the pharmacophore inquiry.The chemical compound that at least a conformation meeting geometric requires is considered to " hitting thing ", and is recorded in the result database.

Other standard is conspicuous to those of ordinary skill in the art, and also can be considered when selection is used for the specific compound of special application, for example the simplicity of chemical constitution, low-molecular-weight, chemical constitution multiformity and water solublity.The application of these standards can be understood well by medical science, calculating and structural chemistry worker.

Obviously, use screening provided herein can optimize compound structure.In these screenings, but the influence that the concrete change of calculated candidate chemical compound produces three dimensional structure, so that optimize the three-dimensional similarity of itself and cyclic peptide.These changes comprise, for example, and the variation of hydrophobicity, spatial volume, electrostatic property, size and bond angle.

Can use the biological test of candidate compound to prove the activity of peptide mimics.Generally speaking, peptide mimics should be to bring into play function to the similar basically mode of the cyclic peptide of structural similarity.In other words, cyclic peptide NAc-CSRRGEC-NH ₂The peptide mimics of (SEQ ID NO:2) should be in conjunction with TRK, and as the use standard in conjunction with the experimental measurement, its affinity is cyclic peptide NAc-CSRRGEC-NH ₂The affinity of (SEQID NO:2) at least half.In addition, use the representative test that the application provides, cyclic peptide NAc-CSRRGEC-NH ₂The peptide mimics of (SEQ ID NO:2) should be regulated the function of TRK-mediation, and its adjusting level is to use N-Ac-CSRRGEC-NH ₂The adjusting level that (SEQ ID NO:2) obtains at least half.

In case the bioactive peptide analogies are identified, use two-dimentional similarity retrieval just can identify relevant analog so.This retrieval can be used, and for example program ISIS Base (Molecular Design Limited) carries out.The two dimension similarity retrieval is allowed other available, closely-related chemical compound of evaluation, and they are easy to screened to optimize biological activity.

6.4 TRK regulator

As noted above, term " TRK regulator " is used for describing any at least one cyclic peptide of the present invention that contains neurotrophin motif RGF (being Arg-Gly-Glu) or molecule of peptide mimics chemical compound of comprising at this.A plurality of cyclic peptide and/or peptide mimics can be present among the regulator of the present invention.In addition, also can comprise extra RGE sequence (for example, connect multiple RGE sequence) in the regulator.

In the Trk regulator, may or may not use joint to separate the RGE sequence, comprise series connection multiple RGE sequence (for example, as in the preferred Trk agonist of the present invention).Joint also can be used for regulator of the present invention is connected on the following solid support or material.

Joint can be any molecule (comprising peptide and/or non-peptide sequence and single amino acids or other molecule), and it does not contain the RGE sequence and energy is covalently bound at least two peptide sequences and/or peptide mimics.Use joint, peptide mimics can be connected with multiple orientation with other peptide or protein sequence.

Joint preferably produces 0.1 to 10 between CAR sequence and/or peptide mimics, the distance between the 000nm, more preferably about 0.1-400nm.Separation distance between the recognition site can be determined according to the desired function of regulator usually.For the Trk antagonist, joint distance should little (0.1-400nm).For the Trk agonist, the joint distance should be 400-10,000nm.A kind of joint that can be used for this purpose is (H ₂N (CH ₂) _nCO ₂H) _mOr derivatives thereof, wherein the scope of n is 1 to about 10, the scope of m is 1 to about 4000.For example, if glycine (H ₂NCH ₂CO ₂H) or its polymer be used as joint; so when using the molecule modeling technique to be connected to other aminoacid; each glycine unit is equivalent to about 2.45 dusts connection distance of 0.245nm in other words, as measuring by the calculating of its low-yield conformation.Similarly, alanine is equivalent to the connection distance of about 3.73 dusts, and aminobutyric acid is equivalent to about 4.96 dusts (connection distance), and aminovaleric acid is equivalent to about 6.30 dusts (connection distance), and aminocaproic acid is equivalent to about 6.12 dusts (connection distance).Other available joint is conspicuous for those of ordinary skills, and comprises, for example based on 2, and the joint of the repetitive of 3-diaminopropionic acid, lysine and/or ornithine.On whether using side chain amino or terminal amino group to decide in the chain link, 2, the 3-diaminopropionic acid can provide the connection distance of 2.51 or 3.11 dusts.Similarly, lysine can provide the connection distance of 2.44 or 6.95 dusts, and ornithine can provide the connection distance of 2.44 or 5.61 dusts.Usually peptide and non-peptide linker can use any proper method well known in the art to be incorporated in the regulator.

The regulator that is the Trk antagonist can contain one or more peptide mimicses.Preferably, these peptide mimicses (promptly not having intervening sequences) adjacent one another are or lean on very closely (just, separate, thereby between peptide mimics, produce about 0.1 distance) to the 400nm scope by peptide and/or non-peptide linker.Obviously, other neurotrophin sequence discussed above is also includable.

As noted above, regulator can be made up of one or more peptide mimicses fully, also can contain extra peptide and/or non-peptide components.Peptide moiety can as indicated abovely synthesize, and perhaps can use recombinant methods.In these methods, all or part of regulator can use the arbitrary multiple expression vector that the definitive host cell is fit to known to a person of ordinary skill in the art to synthesize in living cells.Suitable host cell can comprise antibacterial, yeast cells, mammalian cell, insect cell, plant cell, algae and other zooblast (as hybridoma, CHO, myeloma).The DNA sequence of expressing can the endogenous neurotrophin of coded portion in this way.These sequences can prepare based on known Cdna or genome sequence, or from using the isolating sequence preparation based on the suitable library institute of the probe screening of known cadherin sequential design.Usually these screenings can be as people such as Sambrook, " molecular cloning: laboratory manual " (Molecular Cloning:A Laboratory Manual), Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y., carry out described in 1989 (with the list of references of wherein being quoted).Also can use polymerase chain reaction (PCR), use oligonucleotide primers to separate the nucleic acid molecules of all or part of endogenous neurotrophin of encoding in the method known in the art.For producing the nucleic acid molecules of coding and regulating agent peptide moiety, can use technique known to modify endogenous sequence.Alternatively, the nucleotide sequence of part expectation can use technique known to synthesize, and is joined together to form the sequence of coding and regulating agent part then.

Trk regulator of the present invention can comprise antibody or its Fab of specificity in conjunction with the NT sequence in addition, and perhaps alternatively, specificity is in conjunction with antibody or its Fab of Trk receptor sequence.As used herein, if antibody or its Fab and the peptide that contains NT or Trk sequence are with detectable level (for example, people such as Newton, Develop.Dynamics 1993, in the ELISA reaction that 197:1-13 describes) react, and with the peptide that contains different N T or Trk sequence detectable reaction takes place neither, also not with its in the altered sequence of amino acid residue order in NT (or Trk) and/or the side joint sequence detectable reaction takes place, so, antibody or its Fab are considered to " specificity combination " NT or Trk sequence (having or do not have side joint aminoacid).

Antibody and fragment thereof can use standard technique to prepare.For example referring to Harlow ﹠amp; Lane, " antibody: laboratory manual " (Antibodies:A Laboratory Manual), Cold Spring HarborLaboratory, 1988.In a kind of such technology, originally the immunogen that comprises NT or Trk sequence is injected in arbitrary multiple mammal (mice, rat, rabbit, sheep or goat for instance).Little immunogen (promptly being less than about 20 aminoacid) preferably is connected on the carrier protein, for example bovine serum albumin or keyhole limpet hemocyanin.After the one or many injection, periodically to the animal blood-letting.Can use the regulator or its antigen part that are coupled on the suitable carrier to pass through then, for example, affinity chromatography purification from such antiserum is specific to the polyclonal antibody of NT or Trk sequence.

For example, use Kohler ﹠amp; (" European IMMUNOLOGY KEY WORDS INDEX (Eur.J.Immunol.) 1976, technology 6:511-519) and improvement technology thereof can prepare the monoclonal antibody that is specific to NT (or Trk) sequence to Milstein.Briefly, these methods comprise that the splenocyte preparation that obtains from the animal of immunity same as above can produce the immortal cell line with the specific antibody of expectation.For example, splenocyte by merging with (preferably with immunized animal with pedigree) myeloma cell's fusion partner by immortalization.Select single clone and test the combination activity of their culture supernatant regulator or its antigen part.It is preferred having high response and specific hybridoma.

Can from the supernatant of the hybridoma clone the growth, separate monoclonal antibody, use or do not use different technologies well known in the art to improve its productive rate.By routine techniques, for example chromatography, gel filtration, precipitation and extraction can be removed pollutant from antibody.But the immunofluorescence analysis with the using-system section usually of the active antibody of expectation, cell or localized other sample of target cadherin is identified.

In certain embodiments, monoclonal antibody may be specific to specific NTs, perhaps alternatively, is specific to specific Trk receptor.For example, antibody may be in conjunction with NGF, but not in conjunction with BNDF, perhaps vice versa.As another example, monoclonal antibody possibility specificity is in conjunction with TrkB, but specificity is not in conjunction with TrkA, and perhaps vice versa.The immunity that this antibody can use (producing the antibody at specific NT) to comprise RGE sequence and enough side joint sequences as mentioned above prepared originally, to produce the specificity (for instance, 5 aminoacid of common every side are enough) of expectation.For estimating the specificity of specific antibodies, the antigen that can adopt representative test described herein and/or routine is in conjunction with test.As described herein, these antibody can be used for treatment, diagnosis and test objective usually.For example, these antibody can be connected in medicine and give mammal, thus the Trk-express cell that drug targeting is specific, for example specific neuronal cell.

In certain embodiments, can preferably use antigen-binding fragments of antibodies.These fragments comprise the Fab fragment, and it can use the standard technique preparation.Briefly, can be at protein A pearl post (Harlow ﹠amp; Lane, " antibody: laboratory manual " (Antibodies:A Laboratory Manual), ColdSpring Harbor Laboratory, 1988; See especially page 309) goes up by affinity chromatograph from rabbit anteserum purification immunoglobulin, and produce Fab and Fc fragment by papain digestion.Fab can separate by the affinity chromatograph on protein A pearl post with the Fc fragment.

6.5 the active evaluation of TRK regulator

As noted above, peptide mimics of the present invention, cyclic peptide and other Trk regulator can be regulated the activity of (just, strengthening or inhibition) Trk mediation, for example, comprise neuronal survival, axon growth and synaptic plasticity.Therefore, the active ability of regulator (or doubtful regulator) adjusting Trk mediation can be estimated by measuring one or more these effects in external or the body usually.Generally speaking, in such representative test, if test cell causes measuring the active recognizable destruction that Trk mediates with contacting of material standed for, this test compounds is the Trk antagonist so.In such representative test, if test cell causes measuring the active recognizable enhancing that Trk mediates with contacting of candidate compound, this candidate compound is considered to the Trk agonist usually so.

Particularly in the preferred embodiment of the invention, neurite grows the activity of estimating Trk regulator or candidate compound in the test in vivo.The representative neurite of embodiment grows in the test below one is shown in, and neuron is incubated on the cell monolayer (preferred 3T3 cell or by its deutero-cell line).For example, can set up monolayer 3T3 fibroblast by incubated overnight cell in the single hole of Room 8 hole tissue culture slide (preferably approximately 80,000).Will be from isolating about 3,000 cerebellar neurons of the mouse brain of be born back 3 days (PND3) in control medium (SATO/2%FCS) or replenished on the different monolayers in the culture medium of variable concentrations candidate modulator and cultivated 18 hours.Alternatively, these cells can be incubated in the culture medium of having replenished control peptide (for example, have with Trk regulator cyclic peptide same acid sequence acyclic, linear peptides) or neurotrophin (as NGF, BDNF, NT-3, NT-4, NT-5 or NT-4/5).

Can fix these cell cultures subsequently and carry out GAP43 or with other specificity some reagent dyeings in conjunction with neuron and neurite thereof.Can measure on the male neuron of each GAP43 the length of long neurite then, advantageous applications area of computer aided Morphometry.In this cell culture test, (as suppressing or strengthening) neurite grows to be that the chemical compound of Trk regulator is regulated usually.

6.6 the purposes of TRK receptor modulators

Usually, regulator of the present invention and compositions can be used for regulating (as suppressing or enhancing) by the receptor-mediated activity of Trk, comprise the activity by TrkA, TrkB and/or TrkC mediation.The Trk receptor involves central nervous system's (CNS) growth and reparation, and regulate at least in part resemble that neuronal survival, axon growth, neurite grow, synaptic plasticity and the process of nerve growth one class widely.Therefore, regulator of the present invention and compositions can be used for regulating arbitrary these processes.Wherein, these purposes comprise, are used for the treatment of the situation relevant with these processes, disease and disorderly Therapeutic Method and pharmaceutical composition.Exemplary situation, disease and disorder comprise, only give some instances Alzheimer, parkinson disease, apoplexy, head and spinal cord injury.

In one embodiment of the invention, Trk agonist of the present invention can be used for improving or strengthening by the receptor-mediated activity of Trk.Therefore, Trk agonist of the present invention can be used for, and for instance, improves or strengthen growth and/or the reparation of CNS, for example, by improve or strengthen as neure growth, neuronal survival, axon growth, neurite grow with synaptic plasticity the process of a class realize.Thereby Trk agonist of the present invention can be used for for example treating relate to or the disease and treatment of conditions method relevant with neural injury or impaired function in.Wherein, these comprise above listed disease and disorder.

In other embodiments, Trk antagonist of the present invention can be used for reducing or suppressing by the receptor-mediated activity of Trk.Therefore, the Trk antagonist can suppress as neure growth, neuronal survival, axon growth, neurite grow with synaptic plasticity the process of a class.The Trk antagonist also can be used for for example treating improvement and enhanced Trk receptor active or with the neurotrophin that combines and activate the Trk receptor (for example, in the Therapeutic Method of disease that enhanced activity BDNF) is relevant and disorder (for example, epilepsy).

Also in other embodiments, Trk agonist of the present invention and antagonist also can be used for the reaction that regulate to suppress the CNS growth and repair (i.e. " CNS inhibitor "), comprise inhibition as neure growth, neuronal survival, axon growth, neurite grow with synaptic plasticity the reaction of process of a class.In particularly preferred embodiments, Trk agonist (for example, B of the present invention _AGOr other agonist polypeptide or peptide mimics) can be used for sealing or reduce the reaction of CNS inhibitor.In other embodiments of the present invention, Trk agonist (for example, B _AGOr other agonist polypeptide or peptide mimics) can be used for strengthening and/or promoting neuronic growth and recovery, even suppressing for example administration in the presence of one or more CNS inhibitor of environment.

As special example, be to be understood that to have some inhibitive factor, those inhibitive factor relevant for example with myelin, they can suppress or even stop CNS to grow and the process of reparation, comprise those that exemplify above.The example of these inhibitor includes but not limited to: myelin associated glycoprotein (also being referred to as " MAG "), Nogo-A and oligodendroglia myelin glycoprotein.3.3 top joints are also seen in more complete explanation for these inhibitor.Trk agonist of the present invention and antagonist can be used for regulating the reaction that produces by these and other CNS inhibitor.

Do not really want to be bound by any specific principle or mechanism of action, should be appreciated that the Trk receptor is at least in part by relating to the mechanism adjusting CNS growth and the reparation of protein kinase A (PKA) and phosphoinositide 3-kinase (PI3K).Therefore, in preferred embodiments, Trk agonist of the present invention and antagonist can be regulated the effect of the inhibition signal of being regulated by PKA or PI3K by one or more self that becomes the branch mediation.As an example and unrestricted, the Ser188 of known PKA by direct phosphoric acid Rho molecule activate Rho (people such as Ellerbroek, " journal of biological chemistry " (J.Biol.Chem.) 2003,278:19023-19031).Therefore, Trk agonist of the present invention and antagonist can be used for regulating the signal by the inhibition cascade mediation that relates to Rho.Wherein, these comprise by myelin inhibitor such as MAG (with MAG fusion constructs such as MAG-Fc), Nogo-A, oligodendroglia myelin glycoprotein, NgR, GT1b and 75 ^NTRThe inhibition signal of mediation.Other CNS inhibitor that relates to Rho comprises by the chondroitin sulfate proteoglycan (people such as Monnier from CNS neuroglia cicatrix, " neuroscience " (Neurosci.) 2003,22:319-330) Jie Dao signal, and similarly, these CNS inhibitor also can Trk agonist of the present invention and antagonist adjusting.As another non-limiting instance, the activation of PI3K expection will overcome semaphorins the inhibition activity (people such as Eickholt, " cytobiology magazine " (J.Cell Biol.) 2002,157:211-217).Therefore, Trk agonist of the present invention and antagonist can be used to regulate these CNS inhibitor in addition.

Generally speaking, method of the present invention comprise will express the Trk receptor cell (typically being neuronal cell) in vivo or externally contact with the Trk regulator.The amount of the Trk regulator of administration should be " effective dose ", that is to say, should be the active amount of purpose of effectively regulating the Trk mediation, or alternatively, effectively regulate the amount of purpose CNS inhibitor.In the embodiment of administration Trk regulator as the part of Therapeutic Method, the amount of administration should be situation, disease or the disorderly amount that effective improvement the (but needn't eliminate or cure) treated.Alternatively, the amount of administration can be the amount of one or more symptoms relevant with the situation of being treated, disease or disorder of effective improvement the (but needn't eliminate).

As specific non-limiting instance, Trk regulator of the present invention can be used for regulating (as suppressing or enhancing) nerve growth such as neurite grows.In these methods, neurite grows and can pass through neuron and one or more Trk agonist (cyclic peptide N-Ac-for instance, of the present invention CSRRGELLAASRRGELC-NH ₂) contact and be enhanced and/or instruct.Alternatively, neurite grows and can pass through neuron and one or more Trk antagonisies (cyclic peptide N-Ac-for instance, of the present invention CSRRGEC-NH ₂) contact and be suppressed and/or reduce.The preferred regulator that uses in these methods preferably is connected on polymeric matrix or other holder, and comprises cyclic peptide or its peptide mimics of describing in top 6.1 joints (as describing in 6.3 joints).With or without joint or support material comprise antibody or its segmental regulator also can be used in these methods.

The amount of the method that realization contacts with neuronal cell and the Trk regulator of administration will depend on degree and the character (under the situation of administration Trk antagonist, being degree and the character that expectation suppresses perhaps) that neuronic position and expectation grow.For example, neuron and one or more can be connected to Trk regulator on the support material contact (for instance, by implanting) thus instruct neurite to grow and carry out along support material such as suture, fiber nerve guide wire or other prothesis device.Alternatively, can adopt the hollow zrve guide wire, wherein the nerve guides intracavity contains the compositions that comprises regulator.In vivo, the regulator that can use known technology to implant these nerve guides or otherwise support, thereby, growth and/or treatment spinal cord injury that the neuron that for example promotes to cut off connects.The structure of holder and form should be suitable for the particular injury of being treated, and this is conspicuous for those of ordinary skills.External, polymeric matrix can be used for instructing neuron in the lip-deep growth of expection equally, for example resembles United States Patent (USP) N0.5, as described in 510,628.

6.7 TRK receptor modulators: preparation

In certain embodiments, regulator as described herein is passable, but needn't, be connected in one or more extra molecules.For example, use for some, (it is passable with a plurality of regulators, but needn't be identical) to be connected on holder molecule (as keyhole limpet hemocyanin) or the solid support be favourable, for example (it can be prepared as film or microstructure to polymeric matrix, ultrathin membrane for example), vessel surface (as the surface of tissue culturing plate or the inner surface of bioreactor) or pearl or other granule, they can be by the multiple material preparation that comprises glass, plastics or pottery.Use for some, preferred biodegradable support material, for example cellulose and derivant thereof, collagen protein, the spider's thread or arbitrary multiple polyester (for example derived from hydroxy acid and/or lactone those polyester) or suture (referring to U.S. Patent number No.5,245,012).

Be suitable for regulator is connected to composition and the purpose purposes that method on the support material will depend on holder, and this is conspicuous for those of ordinary skills.Connect and can realize by non-covalent combination usually, for example absorption or affine, or preferably by covalently bound (it can be direct connection between the functional group on regulator and the holder, perhaps can be that the mode with cross-linking agent or joint connects) realization.Regulator can be realized by contact appropriate time with solid support in suitable buffer by the connection of absorption.Change along with temperature time of contact, but usually between about 5 seconds and 1 day, and typically, approximately between 1O second and 1 hour.

Regulator to molecule or solid support covalently bound usually can by at first with support material with have the bifunctional reagent and react realization, this reagent also with regulator on functional group such as hydroxyl, mercapto, carboxyl, ketone group or amino reaction.For example, by the condensation of the aldehyde radical on the holder and amine on the regulator and reactive hydrogen, or the condensation by amino on the holder and the carboxylic acid on the regulator, regulator can be connected to polymerization holder suitable or that use the benzoquinone coating.Producing the preferable methods that connects is by the amino that uses glutaraldehyde.Regulator can be connected on the cellulose by ester bond.Similarly, amido link pair and other molecule keyhole limpet hemocyanin or may be fit to being connected of other support material for example.A plurality of regulators and/or comprise other NT and/or the molecule of Trk receptor sequence can pass through, for example coupling and being connected at random wherein, mixes these molecules of equimolar amounts, and makes its coupling at random with the substrate holder.

Although but regulator preferred combination as described herein arrives on the specific tissue or cell (as neuronal cell and tissue), thereby the site that is enough to expectation in the targeting body for some application, comprises that extra target agent may be favourable.Therefore, the target agent also can, or alternatively, promote the one or more specific tissues of targeting on the regulator thereby be connected to.As used herein, " target agent " can be any material (for example chemical compound or cell), when it is connected on the regulator, promotes the transportation of regulator to target tissue, improves the local concentration of regulator thus.The target agent comprises antibody or its fragment, receptor, part and other is in conjunction with near the molecule target tissue cell or target tissue.Known target agent comprises antibody, agglutinin, adhesion molecule, tumor cell surface binding partner, steroid, cholesterol, lymphokine, haemolysis brinase and those the medicine and the albumen in conjunction with the desired target site of blood plasma hormone, anti-cell surface antigen.The antibody target agent can be complete (whole) molecule, its fragment or its functional equivalents.The example of antibody fragment is F (ab ＇) 2, Fab ＇, Fab and F[v] fragment, they can be produced by conventional method or by gene or protein engineering.Connect covalency normally, and can pass through, directly condensation or other reaction for example, or by two-or many-functional group's joint realize.In other embodiments, with the polynucleotide targeting target tissue of coding and regulating agent, the local concentration that improves regulator thus also is possible.This targeting can use technology well known in the art, comprises that retrovirus and adenovirus infection realize.

For some embodiment, similarly, or selectively medicine to be connected to may be favourable on the regulator.Term used herein " medicine " refers to any bioactivator that is intended to the administration mammal with prevention or treatment disease or other undesirable condition.Medicine comprises hormone, somatomedin, albumen, peptide and other chemical compound.The use of some certain drug is discussed at down in the application's context.

Aspect some, one or more regulators as described herein can be present in the pharmaceutical composition of the present invention.Pharmaceutical composition comprises one or more regulators and one or more pharmaceutically or physiology's associating of going up acceptable carrier, diluent or excipient.These compositionss can comprise buffer (as neutral buffered saline solution or phosphate buffered saline(PBS)), carbohydrate (as glucose, mannose, sucrose or glucosan), mannitol, albumen, polypeptide or aminoacid such as glycine, antioxidant, chelating agen such as EDTA or glutathion, adjuvant (as aluminium hydroxide) and/or antiseptic.And in other embodiments, compositions of the present invention can be formulated into lyophilized preparation.Regulator (separately or with target agent and/or medication combined) is passable, but needn't, use technique known in liposome, to be bundled into capsule.Compositions of the present invention can be formulated as the mode of any suitable administration, for example comprises, local, oral, nose, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration.For some local application, preferably use known composition to be formulated as Emulsion or lotion.

Randomly, pharmaceutical composition is can contain one or more medicines, and these medicines can be connected on the regulator and maybe can be free within the pharmaceutical composition.In fact, be following multiple purpose, any medicine all can be used for multiple use as mentioned below with regulator administering drug combinations as herein described.Can include but not limited to the example of the drug type of regulator administering drug combinations: analgesic, anesthetis, anti-angina pectoris agent, antifungal, antibiotic, anticarcinogen (as paclitaxel or ametycin), anti-inflammatory agent (as cloth Lip river phenol and indomethacin), anthelmintic, antidepressant, antidote, antiemetic, hydryllin, antihypertensive, antimalarial, anti-microtubule agent (as Colchicine or vinca alkaloids), the migraine agent, antimicrobial drug, psychosis, antipyretic, antibacterial, antinoise signal medicine (mobilizing inhibitor) as protease C inhibitor or intracellular calcium ion, antiarthritic, the antithrombase medicine, antitubercular agent, antitussive, antiviral agent, appetite suppressant, the heart medication, chemistry relies on medicine, cathartic, chemotherapeutics, arteria coronaria, brain or peripheral vasodilator, contraceptive, inhibitor, diuretic, expectorant, somatomedin, hormone preparation, sleeping pill, immunosuppressant, narcotic antagonist, parasympathomimetic agent, tranquilizer, analeptic, class sympathetic nerve medicine, toxin (as cholera toxin), tranquilizer and urinary system anti-infective.

Be the imaging purpose, arbitrary multiple diagnostic agent all can be incorporated in the pharmaceutical composition, perhaps is connected on the regulator or is free in the pharmaceutical composition.Diagnostic agent comprises any administration illustrating a certain physiological function in patient's body, and the common impregnable material of other physiological function.Diagnostic agent comprises the enzyme of metal, radiosiotope and radiopaque medium (as gallium, technetium, indium, strontium, iodine, barium, bromine and phosphorus-containing compound), ray agent thoroughly, contrast agent, dyestuff (as fluorescent dye and chromophore) and catalysis calorimetric or fluorescence reaction.Usually, these reagent can use above-mentioned multiple technologies to connect, and can any orientation exist.

Compositions as herein described can be used as a part of administration of extended release preparation (that is to say, for example reach the capsule or the such preparation of gauze of slow release regulator after administration).These preparations can use technique known preparation usually, and by for example, oral, rectum or subcutaneous implantation, or the administration by being implanted in the expection target site.Extended release preparation can contain be dispersed in the carrier matrix and/or be contained in by the speed limit film (for example referring to european patent application 710, the 491A) regulator in around the storeroom.The carrier that is used for these preparations is a biocompatibility, and also can be biodegradable; Preferably, preparation provides constant relatively regulator emission levels.The amount that is contained in the regulator in the extended release preparation depend on implantation site, rate of release and expection the duration and the character of the situation of to be treated or prevention.

The mode administration of the disease that pharmaceutical composition of the present invention can be suitable for (or prevention) to be treated.Proper dosage and persistent period and medicine frequency will be by type and seriousness and and the such factor decisions of medication such as status of patient, patient disease.Usually, proper dosage and therapeutic modality have guaranteed that regulator is to be enough to provide the amount that treats and/or prevents effect.In particularly preferred embodiment of the present invention, regulator as herein described and pharmaceutical composition can be from the dosage range administrations of 0.001 to 50mg/kg body weight on the scheme of odd-numbered day or multiple daily doses, preferably from 0.1 to 20mg/kg.For local application, the Emulsion typical case comprises the amount of the regulator of from 0.00001% to 1% scope, and is preferred 0.0001% to 0.2%, and more preferably from 0.0001% to 0.002%.Fluid composition typically contains about 10ng/ml to 5mg/ml, preferably the peptide mimics from about 10.mu.g to 2mg/Ml.But common service test model of proper dosage and/or clinical trial are determined.Usually the preferred minimum dose that is enough to provide effective treatment that uses.Usually can use a test that is suitable for institute's situation for the treatment of or preventing to come the therapeutic effect of monitored patient, this is well known to those of ordinary skill in the art.

The Trk receptor modulators also can be prepared according to the description that provides in following 6.9 joints.

6.8 p75 bonding agent

Term " p75 receptor-binding agents " is used to describe in the text (as reorganization) natural generation or synthetic molecule, it is in conjunction with the p75 receptor that is combined in the inhibitor complexes, and interference p75 receptor: the interaction of neurotrophin, rather than neurotrophin: the interaction of Trk receptor.Therefore, the p75 receptor-binding agents promotes to suppress neurotrophin-mediated neure growth in the environment.When p75 receptor and nogo receptor and arbitrary myelin associated protein (as MAG, Nogo-A, oligodendrocyte myelin glycoprotein) interaction, it is combined in the inhibitor complexes.The example of p75 receptor-binding agents includes but not limited to: neurotrophin such as NGF, and derived from the reagent of neurotrophin as N-Ac-derived from the NGF coupling collar CTDIKGKEC-NH ₂(SEQ IDNO:43).According to the present invention, if neurotrophin disturb another different neurotrophin in conjunction with the p75 receptor and not with the Trk acceptor interaction that is expressed in the injured neurons surface, so this neurotrophin is the p75 receptor-binding agents.For example, do not express under the neuronic situation of TrkA expressing TrkB, neurotrophin NGF is the p75 receptor-binding agents, because NGF will with the neurotrophin that combines TrkB (as BDNF) competition (promptly disturbing) combination to the p75 receptor, but can not disturb combining of neurotrophin (as BDNF) and TrkB receptor.

In preferred embodiments, the p75 receptor-binding agents comprises at least one contains NGF motif TDIKGKE (being Thr-Asp-Ile-Lys-Gly-Lys-Glu) (SEQ ID NO:42) at its ring ring cyclic peptide or peptide mimics chemical compound.A particularly preferred p75 receptor-binding agents is N-Ac- CTDIKGKEC-NH ₂(SEQ ID NO:43).Mention especially as previous institute, the peptide sequence of line is illustrated in the peptide that passes through the covalent bond cyclisation between the residue of ruling at two ends.In these examples, the p75 receptor-binding agents is by the disulfide bond cyclisation between two cysteine residues, and acetylation is also sealed by amide.Be to be understood that the peptide in conjunction with the p75 receptor preferably will be tied, and therefore be preferably cyclic peptide.The method of peptide cyclisation is described in top 6.1 joints.

A plurality of cyclic peptide and/or peptide mimics can be present in the p75 receptor-binding agents.In addition, extra TDIKGKE sequence (for example, connect multiple TDIKGKE sequence) also can be contained among the p75 receptor-binding agents.

Can or can not utilize joint to separate p75 receptors bind sequence in the p75 receptor-binding agents, the multiple p75 receptors bind sequence of drawing together of bag series connection.Joint can be any molecule (comprising peptide and/or non-peptide sequence and single amino acids or other molecule) that can be covalently attached at least two peptide sequences and/or peptide mimics, and does not contain p75 receptors bind sequence.By using joint, peptide mimics can be connected with multiple orientation with other peptide or protein sequence.

The p75 receptor-binding agents can contain one or more peptide mimicses.Preferably, these peptide mimicses (promptly not having intervening sequences) adjacent one another are or lean on very closely (that is to say, thereby separately between peptide mimics, produced about 0.1 to 400nm distance) by peptide and/or non-peptide linker.The p75 receptor-binding agents can be made up of one or more peptide mimicses fully, perhaps can contain extra peptide and/or non-peptide components.The method for preparing peptide mimics is described in top 6.2 and 6.3 joints.

The all or part of of p75 receptor-binding agents uses the arbitrary multiple expression vector that is suitable for the definitive host cell known to a person of ordinary skill in the art to synthesize in living cells.Suitable host cell comprises antibacterial, yeast cells, mammalian cell, insect cell, plant cell, algae and other zooblast (as hybridoma, CHO, myeloma).The endogenous neurotrophin of DNA sequence codified part of Biao Daing in this way.These sequences can prepare based on known cDNA or genome sequence, or from preparing by the isolating sequence in the suitable library of screening.These screenings usually can be with reference to people such as Sambrook, " molecular cloning: laboratory manual " (Molecular Cloning:A LaboratoryManual), C0ld Spring Harbor Laboratories, Cold Spring Harbor, N.Y., the carrying out of describing in 1989 (with the list of references of wherein being quoted).Also can adopt the polymerase chain reaction (PCR) of using oligonucleotide primers in the method known in the art, separate the nucleic acid molecules of all or part of endogenous neurotrophin of encoding.For producing the nucleic acid molecules of coding and regulating agent peptide moiety, can use known technology to modify endogenous sequence.Alternatively, can use the nucleotide sequence of known technology composite part expectation, be joined together to form the sequence of coded portion p75 receptor modulators then.

6.9 p75 bonding agent: the method that promotes the CNS growth

The invention provides the method that is used to promote the CNS growth, it comprises administration p75 receptor-binding agents.The Trk receptor involve the growth of CNS and reparation and regulate as neuronal survival, axon growth, neurite grow, synaptic plasticity and the process of a class the nerve growth widely.The p75 receptor in conjunction with neurotrophin, and thisly is combined under the situation of p75 receptors bind in inhibitor complexes the ability that the infringement neurotrophin activates the Trk receptor with low-affinity.Therefore, disturb neurotrophin in conjunction with the method for p75 receptor make neurotrophin in conjunction with and activate the Trk receptor, and therefore promote to suppress the neuronic growth of CNS in the environment.

One aspect of the present invention provides a kind of method, and it comprises that the p75 receptor-binding agents that gives the individual treatment effective dose unites at least a neurotrophin.Preferred neurotrophin is NGF, BDNF, NT-3, NT-4 or NT-5.In one embodiment, the p75 receptor-binding agents is to arrive about 100 times amount administration greater than neurotrophin about 10.In another embodiment, the p75 receptor-binding agents is NGF, and neurotrophin is BDNF.Method of the present invention can be used for treating and CNS damage or relevant situation, disease and the disorder of impaired function.Exemplary situation includes but not limited to: Alzheimer, parkinson disease, Heng Tingdunshi family name disease, amyotrophic lateral sclerosis, apoplexy, traumatic brain injury and spinal cord injury.

The method according to this invention, when neurotrophin disturbs another different neurotrophin in conjunction with the p75 receptor that is combined in the inhibitor complexes, and do not disturb this another different neurotrophin when being expressed in the Trk receptor on injured neurons surface, so this neurotrophin is the p75 receptor-binding agents.For example, have the neuronic individuality of expressing the TrkB receptor if NGF and BDNF deliver medicine to altogether, NGF is according to p75 receptor-binding agents of the present invention so, because NGF combines the p75 receptor with the BDNF competition, and does not combine the TrkB receptor with the BDNF competition.

P75 receptor as herein described (combination) agent can be present in the pharmaceutical composition.Pharmaceutical composition comprises p75 receptor-binding agents and one or more pharmaceutically or physiology's associating of going up acceptable carrier, diluent or excipient.These compositionss can comprise buffer (as neutral buffered saline solution or phosphate buffered saline(PBS)), carbohydrate (as glucose, mannose, sucrose or glucosan), mannitol, albumen, polypeptide or aminoacid such as glycine, antioxidant, chelating agen such as EDTA or glutathion, adjuvant (as aluminium hydroxide) and/or antiseptic.And in other embodiments, compositions of the present invention can be mixed with lyophilized preparation.P75 receptor-binding agents (separately or with target agent and/or medication combined) can use technique known to be wrapped into capsule in liposome.

Compositions of the present invention can be formulated as the mode of any suitable administration, for example, comprises part, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration.The pharmaceutical composition that comprises the p75 receptor-binding agents can arrive and in conjunction with the mode administration of the intravital p75 receptor of individual machine with any permission p75 receptor-binding agents.

The sterile injectable form that comprises the pharmaceutical composition of p75 receptor-binding agents can be moisture or butyraceous suspension.These suspensions can use suitable dispersant or wetting agent and suspending agent preparation according to techniques well known.The sterile injectable goods also can be aseptic parenteral solution or the suspensions that is dissolved in nontoxic parenteral acceptable diluent or the solvent.Adoptable acceptable excipient and solvent are sterilized water, lactate Ringer's solution and isotonic sodium chlorrde solution.In addition, aseptic expressed oi is used as solvent or suspension media traditionally.For this purpose, the expressed oi of any gentleness all can be employed, and comprises synthetic list or double glyceride.Fatty acid such as oleic acid and glyceride ester derivatives thereof can be used for preparing injectable medicine, as natural pharmaceutically acceptable oil, and for example olive oil or Oleum Ricini, particularly their polyoxyethylene form.These fluid or suspension also can contain long-chain alcohol diluent or dispersant.The p75 receptor-binding agents also can be prepared according to the description that top 6.7 joints provide.

But p75 receptor-binding agents topical.For example, 75 receptor-binding agents can be applied topically to the individual spinal cord that exposes after spinal cord injury or during the surgical operation.For topical application, pharmaceutical composition can be formulated into the ointment that suitable containing was suspended in or was dissolved in the p75 receptor-binding agents in one or more carriers.The carrier that is used for p75 receptor-binding agents topical includes but not limited to: mineral oil, liquid petrolatum, white petrolatum, emulsifing wax, water or the absorbable material of type i collagen gel or the such class of gelatin hemostatic gauze for example, (Gelfoam , Pharmacia ﹠amp; Upjohn, Kalamazoo, MI).

Suitable dosage and persistent period and frequency are decided by more such factors: patient's situation, the type of patient disease and seriousness and medication.Usually, proper dosage and therapeutic modality guarantee that the p75 bonding agent is enough to provide the amount that treats and/or prevents effect.Be used for determining that the various considerations of suitable dose are described in, for instance, Gilman etc. (editor), " pharmacological basis of treatment " (The Pharmacological Bases of Therapeutics), the 8th edition, (1990), PergamonPress.Proper dosage can adopt experimental model and/or clinical trial to determine usually.Usually the preferred lowest dose level that is enough to provide effective treatment that uses.The curative effect to the patient is monitored in the test of using to health check-up, imaging research well known to those of ordinary skill in the art or being suitable for the disease that institute treats or prevent.Adjust dosage based on monitoring result.For example, after giving, can monitor the recovery of the individual arm consciousness that has the spinal cord injury relevant by health check-up with the arm unconsciousness according to p75 receptor-binding agents of the present invention.

The compositions that comprises the p75 receptor-binding agents can be used as the part of extended release preparation (just, for example reaching the capsule or the such preparation of gauze of slow release p75 receptor-binding agents after administration) by administration.These preparations can use technique known to be produced usually, and by for example subcutaneous implantation or be implanted in the target site administration of expection.Extended release preparation can contain be dispersed in the carrier matrix and/or be contained in the speed limit film (for example referring to european patent application 710,491A) the p75 receptor-binding agents in around the storeroom.The carrier that is used for these preparations is a biocompatibility, and also can be biodegradable; Preferably, preparation provides constant relatively bonding agent emission levels.The release duration that the amount that is contained in the bonding agent in the extended release preparation depends on implantation site, rate of release and expectation is the character of situation to be treated.

But although p75 receptor-binding agents preferred combination as herein described in specific tissue or cell (being neuronal cell and tissue), thereby the site that can be enough to expectation in the targeting body use for some, comprise that extra target agent may be favourable.Therefore, the target agent can be connected to the p75 receptor-binding agents to promote one or more specific tissues of targeting.As used herein, " target agent " can be any material (for example chemical compound or cell), when it is connected on the p75 receptor-binding agents, promote of the transportation of p75 receptor-binding agents to target tissue (as injured neurons), improve the local concentration of p75 receptor-binding agents thus.

The target agent can comprise antibody or its fragment, receptor, part and other is in conjunction with near the molecule target tissue cell or target tissue.Known target agent comprises antibody, agglutinin, adhesion molecule, tumor cell surface binding partner, steroid, cholesterol, lymphokine, haemolysis brinase and those the medicine and the albumen in conjunction with the desired target site of blood plasma hormone, anti-cell surface antigen.The antibody target agent can be complete (whole) molecule, its fragment or its functional equivalent body.For example, MAG, Nogo-A or myelin glycoprotein antibody can be the target agent.The example of antibody fragment is F (ab ＇) 2, Fab ＇, Fab and F[v] fragment, they can be produced by traditional method or by gene or protein engineering.Connect covalency normally, and can be by for example directly condensation or other reaction, or by two-or many-functional group's joint realization.In other embodiments, with the polynucleotide targeting target tissue of coding and regulating agent, the local concentration that improves regulator thus also is possible.This targeting can use technology well known in the art, comprises that the infection of retrovirus and adenovirus realizes.

For some embodiment, it is favourable that medicine is connected on the p75 receptor-binding agents.To the description of the medicine that be fit to connect the p75 receptor-binding agents referring to top 6.7 joints.

7. embodiment

The present invention also passes through the following example to describe and demonstration.Yet, in the description herein and the application of other local embodiment only be illustrative, and do not limit the present invention in any way or the scope and the implication of any illustration term.Similarly, the present invention's any certain preferred embodiment of being not limited to describe herein.In fact, after having read this description, many modifications and variations of the present invention all are conspicuous for those skilled in the art, and can carry out these variations and can not depart from the spirit or scope of the present invention.Therefore the present invention is only with the term of appended claims and be equal to all scopes that these claim given and exceed.

7.1 experimental procedure

7.1.1 neurite grows test

With people such as previous Williams (" neuron " (Neuron) 1994,13:583-594) method of Miao Shuing is at monolayer parent 3T3 cell or LK8 cell (the transfection 3T3cells of the chicken N-cadherin of the expression physiological level of establishment; Referring to people such as Doherty, " neuron " (Neuron) 1991 set up cerebellar neuron on 6:247-258) and cultivated altogether.Cell maintains in the DulbeccoShi improvement EagleShi culture medium of having replenished 10% hyclone (FCS).

For setting up coculture, in about 80,000 3T3 cells (or LK8 cell) coated plate each chamber in the eight Room tissue culture slides that are coated with poly-L-Lysine and fibronectin.The coated plate cell spent the night maintain in the DulbeccoShi improvement EagleShi culture medium of having replenished 10%FCS, to form confluent monolayer.Remove culture medium and will about 6,000 dissociated cerebellar neurons (taking from the back 9 days rat that is born) coated plate in each hole that has replenished 2%FCS SATO culture medium.Adding test agent as described herein, and keep common cultivation 18 hours.Then coculture is fixed and the dyeing of GAP-43 immunoreactivity.Neuron between about 120 to 150 is measured the average length of the longest neurite of each cell, equally as people such as Williams (" neuron " (Neuron) 1994,13:583-594) before described.

7.1.2 the molecule modeling of Trk receptor-ligand structure

Use the X ray crystalline texture of NGF/TrkA and NT-4/TrkB complex to carry out the molecule modeling.These structures were before existing to be described (respectively referring to, people such as Wiesmann " nature " (Nature) 1999,401:184-188; And people such as Banfield, Structue (Camb) 2001,9:1191-1199), and can, for example, obtain easily with accession number 1www (being used for NGF/TrkA) and 1hcf (being used for NT-4/TrkB) by Protein Data Bank (PDB) network.The structure of using Swiss PDB software kit to separate the different motifs that derive from the crystal combination contact surface uses Accelrys to produce image.

Can be based on part residue sequence number, by the receptor contact average of each part residue in measuring, by the various ligand/receptor contact surfaces generation contact types of NT-4/TrkB crystal structure.Per three residue forms obtain an average, and contact number is that given part aminoacid five dusts are with interior receptor residue number.

7.1.3 reagent

Recombined human FGF2, BDNF and NT-4/5 are all available from R﹠amp; D system (Minneapolis, MN).CB1 cannabis receptor stimulating agent WIN55, the acquisition of 2122-2 mesylate and use as previously mentioned (referring to, people such as Williams, " cytobiology magazine " (J.Cell.Biol.) 2003,160:481-486).Trk receptor antagonist K252a available from Calbiochem (San Diego, CA).PD173074 FGFR antagonist (people such as Mohammadi, Embo J 1998,17:5986-5904) as people such as previous Skaper (" neuro chemistry magazine " (J.Neurochem.) 2000,75:1520-1527) and people such as Hamby, " journal of medicinal chemistry " (J.Med.Chem.) 1997,40:2296-2303) described synthetic and use.

Synthetic peptide all available from commercial supplier (Multiple Peptide Systems, San Diego, CA).All peptides use conventional method with reversed phase high-performance liquid chromatography (RP-HPLC) purification, and obtain (promptly pure greater than 95%) with the purity of top level.Pass through the peptide of covalent bond cyclisation between the residue of underlined peptide sequence (running through whole description) expression with two terminal leukorrhagia line.In these embodiments, peptide by the disulfide bond between two cysteine residues by cyclisation, acetylation and carry out amide sealing.

7.2. evaluation with the NT-4 linear peptides sequence of TRKB acceptor interaction

This section has been described from testing in conjunction with the molecule modeling of identifying suitable linear peptides (LIP) sequence the natural neurotrophin part of Trk receptor critical sites.

Considerable evidence shows that nearly membrane immunoglobulin (Ig) domain (D5) of Trk receptor participates in the combination of NTs directly.Referring to, for example, people such as Perez, " molecular cell neuroscience " (Mol.Cell Neurosci.) 1995,6:97-105; And people such as Urfer, Embo J.1995,14:2795-2805.NGF/TrkA complex (people such as Wiesmann, " nature " (Nature) 1999,401:184-188) and NT-4/TrkB complex (people such as Banfield, Structure (Camb) 2001, crystal structure 1191-1199) is resolved, and it supports this viewpoint.In two kinds of structures, single NT dimer is attached in the Trk acceptor molecule, and wherein each the NT molecule in this dimer combines with each Trk acceptor molecule successively.

Use the algorithm of design to analyze two kinds of crystal structures, with the range of linearity of the part of labelling and receptors bind (people such as Doherty, " molecular cell neuroscience " (Mol.Cell Neurosci.) 2000,16:283-295).In order to demonstrate this analysis, NT-4/TrkB (D5) crystal structure is shown in Fig. 6 A at this.In this complex, independent NT monomer (is labeled as a ₁) contact with two TrkB acceptor monomers (being labeled as b1 and b2) are linear.These two contact surfaces are analyzed, and this analysis chart is shown in Fig. 6 B-6C.In these figure, the contact surface between NGF and TrkA receptor dots, and the contact surface between NT-4 and the TrkB is represented with solid line.To the observation of this two figure as can be seen, the contact surface between these ligand-receptor complex is obviously overlapping.

The hookup type analysis shows that the N-end of NT part contacts the tightst (Fig. 6 C) with the Trk receptor.In addition, a little linear motif (SRRGE) that is positioned at contact pattern main peak exists with half spiral status and can be considered as a sealing ring.This sequence very conservative in BDNF (ARRGE) (it also with TrkB receptors bind), and among the NGF of the part that is respectively TrkA and TrkC receptor (FHRGE) and NT-3 (SHRGE), partly guard.What is interesting is, this zone of neurotrophin in crystal structure not be in conjunction with NT unordered (referring to, people such as McDonald, " nature " (Nature) 1991,354:411-414) and therefore before do not become the theme of peptide research.

a ₁/ b ₁Contact surface contact pattern is set forth in Fig. 6 B.NT ring 1-4 is labeled, other people think they involve NT/Trk interact (people such as LeSauteur, " journal of biological chemistry " (J.Biol.Chem.) 1995,270:6564-6569).Yet neither one appears at a in these rings ₁/ b ₁Contact surface has only ring 1 to participate in a ₁/ b ₂Contact surface.

7.3 the exploitation of TRKB antagonist peptide

With separate cerebellar neuron from the PND2 rat pup be incubated at control medium or replenished the BDNF of finite concentration scope and/or the monolayer 3T3 fibroblast in the NT-4 culture medium on.After 16 hours, fixedly coculture measure as previously mentioned neurite average length (people such as Williams, " journal of biological chemistry " (J.Biol.Chem.) 2000,275:4007-4012).Be set forth in result among Fig. 7 A and show two kinds of parts all with the prominent growth of exciting nerve of the mode of dose dependent, wherein maximum reaction see about 1 and 10ng/ml between.

Above result in the 7.2 joints suitable constraint peptide that shows the little linear RGE motif that is present among all NTs and natural NT structure should to have structure highly overlapping, and have the function of Trk receptor antagonist thus.For verifying this hypothesis, designed a kind of LIP of annular form, it is by the disulfide bond constraint and have aminoacid sequence N-Ac- CRGEC-NH ₂Grow at above-mentioned neurite and to have tested the effect of this peptide to BDNF and NT-4 reaction in the test, wherein the working concentration of two kinds of NT parts is 5ng/ml.Be set forth in these results among Fig. 7 B, show this peptide antagonism BDNF and NT-4 reaction, wherein during 144 ± 23 μ M to the suppression ratio of BDNF reaction and when 112 ± 22 μ M suppression ratio to the NT-4 reaction be 50%.On the contrary, when test concentrations surpassed 400 μ M and lack any natural NT part, peptide did not have effect for growing of basic neurite.These results show that cyclic peptide itself does not have special effect for growing of neuronic survival and neurite.

7.4.NT-4 LIP is than NT3 equivalents and the stronger TRKB inhibitor of NGF LIPS

All NTs enjoy the RGE sequence motifs.Yet the amino sequence of this motif flank is different between TrkA, TrkB and TrkC.Therefore, tested a series of " being equal to " peptide, to determine whether the extension peptide from the TrkB part is a kind of more activated TrkB acceptor inhibitor by the sequential design of different N T part.

The molecule Modeling Research shows peptide N-Ac- CSRRGEC-NH ₂To have structure overlapping with the natural SRRGE motif of NT-4.Correspondingly, grow at the described neurite of above-mentioned 7.3 joints and tested this peptide sequence in the experiment together with derived from NGF (N-Ac- CFHRGEC-NH ₂) and NT-3 (N-Ac- CSHRGEC-NH ₂) be equal to the ability that peptide suppresses BDNF and NT-4 reaction.Surprisingly, derived from the N-Ac-of NT-4 CSRRGEC-NH ₂Be better than about 5 times of NGF and NT-3 derived peptide aspect the inhibition BDNF reaction, wherein the suppression ratio when 27 ± 6 μ M is 50%.These results are illustrated in Fig. 7 C.

As noted above, add fewly making peptide reach 5 times to two side joint amino acid residues at the effect increase of the reaction of TrkB from NT-4.Interpolation does not have a bit effect from the aminoacid that is equal to of NGF or NT-3 to original ring-type RGE peptide, shows that the bonded selectivity of NT can be at least in part decided by the character of the amino acid residue of direct side joint RGE motif.In fact, considerable evidence shows that the specificity of Trk receptors bind is by the sequential coding of NTs amino terminal.Referring to, for example, people such as Urfer, Embo J.1994,13:5896-5909; And McInnes ﹠amp; Sykes, " biopolymer " (Biopolymers) 1997,43:339-366.These discoveries show that cyclic peptide of the present invention and peptide mimics can be by selecting from the RGE side joint aminoacid sequence of the NT part of preferred combination desirable T rk receptor and the specific Trk receptor (for example, targeting TrkA, TrkB or TrkC receptor) of targeting.

When peptide being tested, can see the reaction (referring to, Fig. 7 D) of character same as described above with regard to NT-4.Yet, though the peptide N-Ac-of only about 25 μ M CSRRGEC-NH ₂Just can obtain the inhibition of 50% pair of BDNF reaction, but the NT-4 of same level reaction need to suppress the peptide of about 55 ± 4 μ M.

As cyclic peptide N-Ac- CRGEC-NH ₂Like that, N-Ac- CSRRGEC-NH ₂Peptide or its NGF or NT-3 equivalents all grow without any influence basic neurite in the control medium of not replenishing the NT part.Also having tested these peptides suppresses by other reagent-comprise that the neurite of N-cadherin, FGF2 or CB1 receptor stimulating agent-stimulation grows the ability of reaction.The cell growth response that produces by these reagent place in the elsewhere existing the description (people such as Williams, " cytobiology magazine " (J.Cell Biol.) 2003 160:481-486), particularly, is considered to not comprise the Trk receptor.Those experimental results are shown in Fig. 8.Particularly, even give with the concentration that suppresses NT-4 and BDNF reaction fully, these cyclic peptide do not suppress any of these reaction yet.These data acknowledgements cyclic peptide of the present invention and peptide analogues can suppress the Trk function of receptors fully, and neurite is grown without any nonspecific effect.

Also grow and estimated linear peptides N-Ac-SRRGELA-NH in the test at neurite at NT-4, BDNF and above mentioned other reagent ₂Effect.These results also are shown in Fig. 8.As expected, although the annular form of this peptide is the potent inhibitor of NT-4 and BDNF reaction, even when concentration brought up to 125 μ M test, linear peptides does not suppress any reaction yet.Therefore, the peptide and the peptide mimics needs that contain the RGE motif for example retrain by disulfide bond, just can become functional Trk receptor antagonist.

7.5.TRKB the exploitation of agonist peptide

In the crystal structure of NT-4/TrkB receptor complex, self arranged anti-parallel in the NT-4 dimer of the SRRGE motif among the NT-4.Corresponding motif shows similar arranged anti-parallel in the crystal structure of NGF/TrkA receptor complex.Before, used " series connection repeats " simulation method to develop the peptide agonists of N-cadherin.Referring to, people such as Williams, " journal of biological chemistry " (J.Biol.Chem.) 2002,277:4361-4367.The arranged anti-parallel of RGE motif shows that " series connection repeats " method also can be used to develop the Trk receptor agonist peptides in the neurotrophin.

The hypothesis that molecule modeling support is such, the multiple NT-4SRRGEL sequence of promptly connecting can be constrained on cyclic peptide N-Ac-in conjunction with the mode of two TrkB acceptor monomers to allow the while CSRRGELAASRRGELC-NH ₂(this peptide also is referred to as " B in the text _AG" peptide) in.The B of outstanding this point _AGThe model structure of peptide is shown in Fig. 9 in the literary composition.Therefore, tested B in the test of the description of 7.3 joints in the above _AGThe influence that peptide grows neurite.

This experimental result is shown in Figure 10 A.Can see that this peptide is with the mode of dose dependent excite nerve prominent growing, wherein EC ₅₀About 300nM, and when about 600Nm, approach maximum reaction.As the result of native ligand BDNF and NT-4, neurite grows B _AGThe reaction of peptide is two stage (comparison diagram 7A and 10A).Next, compare B _AGPeptide and fixed growth peptide are to the stimulation ability of neural axon growth.These experimental datas are shown in Figure 10 B, show the B of 6 μ M _AGPeptide promotes axon growth with the degree identical with the maximum activated concentration of NT-R, BDNF and FGF2.

7.6 the TRKB antagonist suppresses the agonist peptide reaction

In order to verify B _AGPeptide is carried out experiment to determine peptide antagonists N-Ac-by activate Trk receptor (on be set forth in the 7.3-7.4 joint) in conjunction with the site identical with the monomeric peptide antagonist CSRRGEC-NH ₂Whether can suppress B _AGThe effect that peptide grows neurite.The results are shown in Figure 11.

At 125 μ M, this TrkB antagonist Toplink suppresses B fully _AGThe activity of peptide under maximum activated concentration.On the contrary, the linear forms of this peptide (are peptide N-Ac-SRRGELA-NH ₂) grow in the test almost to B at neurite _AGThe activity of peptide is influence not.K252a a kind ofly it is reported that (people such as Tapley, " proto-oncogene " (Oncogene) 1992 7:371-381), suppresses neurite equally fully and grow B into the chemical compound of the Trk receptor antagonist of relative specificity _AGThe reaction of peptide.Yet, PD17304, a species specific FGF receptor antagonist does not suppress this reaction.

These data show, are specificity and effective Trk receptor antagonist based on " series connection repeats " cyclic peptide of RGE motif and peptide mimics.

7.7.TRK agonist is restrained the inhibitor of neure growth

This embodiment has described research Trk receptor stimulating agent and has suppressed the additional experiment of the effect that had under the condition of neure growth usually.Especially, these experiments show, are different from natural Trk receptors ligand, and Trk receptor stimulating agent of the present invention can resist the activity of inhibitor molecules and/or its receptor.

7.7.1 material and method

Reagent and cultivation are handled.Except as otherwise noted, obtain in the same 7.1.3 joint of the mentioned experiment reagent of this section and the following chapters and sections and set forth.Especially, recombined human FGF2 and BDNF are available from R﹠amp; (Minneapolis MN), and uses with final concentration 5ng/ml d system.(San Diego CA), and uses with final concentration 100nM Trk receptor stimulating agent K252a available from Calbiochem.Trk agonist peptide B _AG(SEQ ID NO:18) is available from commercial supplier (Multiple Peptide Systems, San Diego CA).Reorganization MAG-Fc chimera is available from R﹠amp; (Minneapolis MN), and uses with final concentration 5-25 μ g/ml d system.(Falmouth MA), and uses with final concentration 20 μ g/ml the monoclonal antibody of GTlb (clone GMR5) available from Seikagaku America.P75 ^NTRRabbit polyclonal antibody is as discussed previously, be anti-this receptor ectodomain produce (see Huber﹠amp; Chao, " developmental biology " (Dev.Biol.) 1995 167:227-238), and used with 1: 200 serum dilution.(SanDiego CA), and uses with final concentration 200 and 400nM respectively available from Calbiochem for known PKA inhibitor KT5720 and H-89.(San Diego CA), and all uses with final concentration 10 μ M available from Calbiochem equally for known PI3K inhibitor Wortmnannin and LY294002.(Bristol UK), and uses with final concentration 10 μ M Rho inhibitors of kinases Y27632 available from Tocris.

All reagent are being diluted in the culture medium altogether, and joining in the culture before being normally just at the neuron coated plate.Exception is anti-p75 ^NTRThe antiserum that receptor produces.Alternatively, high density neuron suspension was handled 60 minutes with 1: 200 serum dilution.Dilute about 20 times of neuron then, and plantation is cultivated.P75 in the culture ^NTRThe residual quantity of antibody estimates to be about 1: 5000 serum dilution.The separate control experiment shows that this antibody grows not influence to neurite when 1: 1000 dilution factor, prove that 1: 5000 used in this experiment dilution factor has negligible effect at the most.

Neurite grows test.Neurite grows test as the operation of preceding 7.1.1 joint.

7.7.2 result

Trk receptor stimulating agent B _AGSealing MAG suppresses active.Before shown when myelin associated glycoprotein (MAG) as cytostromatic transfection molecule (people such as Mukhopadhyay, " neuron " (Neuron) 1994,13:757-767) present or as solubility Fc chimeric protein (people such as Tang, " molecular cell neuroscience " (Mol.Cell.Neurosci.) 1997 9:333-346) adds the reaction that grows that can suppress neurite when giving birth back 2-3 days rat cerebellum neuron.For going deep into these research, the back 3 days cerebellar neuron of birth is incubated on the monolayer LK8 cell, this cell is a kind of 3T3 fibroblast that can express transfection N-cadherin, and what before shown that it can promote neurite powerfully grows reaction (people such as Williams, " neuron " (Neuron) 1994,13:583-594).The concentration that exists in cell and the culture medium is that the warm albumen of the soluble M AG-Fc of 0,5 or 25 μ g/ml is cultivated together.Just as was expected, and MAG-Fc suppresses growing of neurite in the mode of dose dependent, and the suppression ratio when the concentration in the culture medium is 25 μ g/ml is about 40% (referring to Figure 12).

Previous report shows, inhibitor such as MAG are by its effect that RhoA and/or the kinase whose activation of the sub-Rho of its downstream effect are regulated them.Referring to, for example, people such as Dergham, " Journal of Neuroscience " (J Neurosci.) 2002,22:6570-6570; People such as Fournier, " Journal of Neuroscience " (J Neurosci.) 2003,23:1416-1423; And people such as Lehmann, " Journal of Neuroscience " (J Neurosci.) 1999,19:7537-7547.In order to verify these reports, also be that the known Rho inhibitors of kinases Y27632 of 10 μ M is incubated at together (people such as NaruMiya, " Enzymology method " (Methods Enzymol.) 2000,325:273-284 with cell and concentration; People such as Davies, " journal of biological chemistry " (Biochem.J.) 2000 is 351:95-105) in the culture medium.As expected, under these conditions, MAG-Fc does not suppress growing of neurite, even its concentration in culture medium also is so (Figure 12) during up to 25 μ g/ml.

Also having carried out the another one neurite grows experiment and may what effect be arranged to inhibitor such as MAG with research (if any) Trk agonist.In these experiments, be the Trk inhibitor polypeptide B of 6 μ M with the concentration that exists in neuron and the culture medium _AG(SEQ ID NO:18, aforementioned in 7.5 joints) cultivates together.Surprisingly, MAG-Fc does not suppress growing of neurite under these conditions, even its concentration in culture medium also is so (Figure 12) during up to 25 μ g/ml.On the contrary, when neurotrophin BDNF is present in the culture medium with 5ng/ml concentration, without any measurable effect-that is to say that MAG-Fc continues to suppress neurite and grows (Figure 12) to MAG reaction.These results conform to previous report: promptly neuronal cell must come " sensitization " with neurotrophin, thus stop MAG and myelinic inhibitor activity generation (referring to, people such as Cai, " neuron " (Neuron) 1999,22:89-101).

In order further to study B _AGThe ability of polypeptide sealing MAG inhibitor activity, neurite grow test with various different branch culture medium concentration determinations this polypeptide.These experimental results are depicted in Figure 13.These data show when the concentration in the culture medium is low to moderate 30nM, B _AGPolypeptide has effectively sealed the activity of MAG inhibitor, its concentration about 100 and 200nM between the time reach the maximum reaction of half.

In order to confirm that these experimental results are not is that to suppress any specific MAG of N-cadherin composition in being grown by neurite caused, has also used to cultivate and has tested at the neuron of not expressing on the monolayer 3T3 fibroblast of transfection N-cadherin.The bar chart that shows these experimental datas is seen Figure 14.Though when cell culture during in these conditions basic neurite to grow reaction lower, concentration is that the MAG-Fc of 25 μ g/ml has still produced neurite is grown inhibition that can survey, substantial.When not having MAG-Fc, the basic horizontal that neurite grows is vigorous already, and when the concentration in the culture medium is 6 μ M B _AGPolypeptide does not have substantial effect.Yet, observe Figure 14, show that the Trk-receptor stimulating agent has sealed the MAG reaction really effectively under this concentration, thereby MAG-Fc can not suppress neurite and grows when final concentration is 25 μ g/ml.As preceding, again with B _AGEffect opposite, when concentration was 5ng/ml, neurotrophin BDNF did not have obvious effects to the inhibitory reaction that is stimulated by MAG-Fc.

These experiments show Trk receptor stimulating agent such as B _AGPolypeptide can be used for effectively preventing or reduces the inhibitory reaction that signal transduction molecule produced by for example MAG.These experimental results also show Trk receptor stimulating agent (B for example _AG) promote neuronic growth and recovery, even when also being like this during administration in the inhibition environment that is for example suppressing signaling molecule MAG existence.

B _AGSealing is by the inhibition of GT1b.B _AGPolypeptide stops GT1b to suppress active ability also to grow in the test at neurite and inquire into.Previous report described the GT1b multivalent antibody IgM that the neurite that can suppress to come from cerebellar myeloid grows (people such as Vinson, " journal of biological chemistry " (J.Biol.Chem.) 2001,276:20280-20285).In order to confirm these reports, cultivate cerebellar neuron on control medium and the monolayer that contains in the culture medium of 20 μ g/ml GT1b monoclonal antibodies is expressed on the 3T3 cell of N-cadherin.These experimental datas are shown on the bar chart of Figure 15.Consistent with previous report, suppressed neurite growing under this condition strong with 20 μ g/ml antibody co-cultured cells.Relatively Figure 15 center pillar is marked the post C among (1) and this figure.Eliminated this effect effectively with 10 μ MRho inhibitors of kinases Y27632 co-cultured cells, the GT1b receptor that has confirmed previous report relate to the Rho kinases as downstream effect of its signal cascade (referring to, people such as Vinson, " journal of biological chemistry " (J.Biol.Chem.) 2001,276:20280-20285).Surprisingly, as Trk-receptor stimulating agent B _AGWhen the antibody of (6 μ M) and GT1b was present in the culture medium simultaneously, the inhibitor effect of antibody had effectively been removed; Promptly observe with culture medium in the neurite of being on close level when not having antibody grow.Relatively Figure 15 center pillar is marked the post C among (2) and this figure.

These results show Trk receptor stimulating agent such as B _AGPolypeptide can be used for effectively preventing or reduces the inhibitory reaction that receptor produced by for example GT1b.These experimental results also show Trk receptor stimulating agent (B for example _AG) promote neuronic growth and recovery, even administration also is like this when suppressing in the environment, for example suppressing signaling molecule MAG to exist.

B _AGSealing p75 ^NTRInhibition.Owing to think that the inhibition molecule in the myelin is situated between directly or indirectly by p75 ^NTRThe receptor conducted signal is so also studied B _AGThe prevention this receptor of peptide suppresses active ability.At first, suppress growing of neurite really, cerebellar neuron is incubated at control medium and contains p75 in order to verify signal conduction from this receptor ^NTRMonolayer in the culture medium of polyclonal antibody (serum dilution in 1: 200) is expressed on the 3T3 cell of N-cadherin.

These experimental datas are shown in the bar chart of Figure 16.Effectively suppressed neuronic subsequently growth in 60 minutes with the antibody pretreatment cell, this point can be by visual comparison Figure 16 bar chart center pillar mark (1) and C as seen.As MAG and GT1b, after antibody treatment, Rho inhibitors of kinases Y27632 is added in the neuron p7 immediately with final concentration 10 μ M ^NTRAntibody do not cause inhibitory reaction (referring to Figure 16 post (2)).Similarly, with final concentration be the B of 6 μ M _AGPolypeptide is cultivated neuron also can effectively seal p75 ^NTRThe inhibition effect of antibody.Yet with neurotrophin BDNF (final concentration is 5ng/ml) cultured cell to by p75 ^NTRThe inhibitory reaction that antibody causes does not have obvious effects.

Because handle the antibody (estimation is no more than about 1: 5000 serum dilution) that the back cell culture may contain some residual quantities, cultured cell carries out controlled trial in the culture medium of the polyclonal antibody of 1: 1000 serum dilution.The existence of this horizontal antibody grows neurite does not have measurable influence, shows that these test viewed effect is not caused by handling the residual extremely low-level antibody in back.

These experimental results show Trk receptor stimulating agent such as B _AGPolypeptide can be used for effectively reducing or stoping p75 ^NTRThe inhibitory reaction that approach produced.This result also shows Trk receptor stimulating agent (B for example _AG) promote neure growth and recovery, even when suppressing for example there is p75 in the environment ^NTRWhen conduction administration of inhibition signal also be like this.

B _AGThe signal conduction is by PKA and PI3K mediation.For further research Trk receptor stimulating agent sealing suppresses the mechanism of signal, with cerebellar neuron in control medium or replenished through being defined as the B of maximum activated concentration _AGThe 3T3 monolayer culture of the culture medium of polypeptide (6 μ M final concentration), neurotrophin BDNF (5ng/ml final concentration) or FGF2 (5ng/ml final concentration) 18 hours.These experimental results are described in the bar chart with Figure 17.Under these conditions, the three kinds of factor (B _AG, BDNF and FGF2) each comparison compare the about 60-70% of the neurite lengths that all extended according to cultivating.When with K252a, (people such as Tapley, " proto-oncogene " (Oncogene) 1992 is when 7:371-381) adding culture medium with the final concentration of 100nM, by B for a kind of Trk receptor antagonist that is reported as relative specificity _AGSubstantially eliminate with the reaction that grows that BDNF produces.Yet, unaffected by the growth response that FGF2 produces, confirmed such report, be that FGF2 is by being different from the Trk receptor, and the signal transduction cascade that does not particularly comprise PKA or PI3K promotes that neurite grows (referring to people such as Williams, " cytobiology " (Cell Biol.) 2003,160:481-486).

In similar experiment, with cell and protein kinase A (PKA) inhibitor KT5720 (200nM final concentration) or H-89 (400nM final concentration) or phosphoinositide 3-kinase (PI3K) inhibitor Worthmannin (10 μ M final concentration) or LY294002 (10 μ M final concentration) cultivation together in culture medium.The same with the Trk receptor antagonist, the neurite of BAG and BDNF is grown reaction eliminated substantially by these inhibitors of kinases.As expected, it is unaffected the neurite of FGF2 to be grown reaction.

These results show that activated Trk receptor is by the activation mechanism that relates to PKA and PI3K prominent the growing of exciting nerve.Therefore, Trk agonist of the present invention (B for example _AGPolypeptide) can seal or reduce the various inhibition signals of vast scope effectively.Especially, Trk agonist of the present invention can stop the inhibition signal of being made up of the one-tenth branch of PKA or PI3K inhibition or inactivation one or more that signal cascade mediated effectively.

For instance and and limit, PKA it is reported by the Ser188 on the direct phosphorylation Rho come this molecule of inactivation (people such as Ellerbroek, " journal of biological chemistry " (J.Biol.Chem.) 2003,278:19023-19031).Therefore, Trk agonist of the present invention can be used for the signal that seals or reduce to be mediated by the inhibition cascade that relates to Rho.Wherein, these comprise by myelin inhibitor for example MAG (or by MAG fusant for example MAG-Fc), Nogo-A, oligodendrocyte myelin glycoprotein, NgR, GT1b and p75 ^NTRThe inhibition signal of mediation, and by from the signal of the chondroitin sulfate proteoglycan of CNS neuroglia cicatrix mediation (people such as Monnier, " neuroscience " (Neurosci.) 2003,22:319-330).As another limiting examples, the activation of PI3K expection with the inhibition activity of restraint signal element (people such as Eickholt, " cytobiology magazine " (J.CellBiol.) 2002,157:211-217).In fact, neurotrophin it is reported by activate Trk-PI3K cascade in the neuron restrains the inhibition signal (people such as Atwal, " Journal of Neuroscience " (J.Neurosci.) 2003,23:7602-7609).Therefore, Trk agonist of the present invention also can be used for sealing or reduces these suppressing signal.

7.8. extra TRK agonist compound

Present embodiment described extra based on or derived from B in the previous embodiment _AGThe peptide of polypeptide and peptide mimics.The biological test data also are provided, have proved that these new compounds also show the activity as the Trk receptor stimulating agent.

7.8.1 novel Trk receptor stimulating agent

Following peptide and peptide mimics are based on aforesaid B _AGAmino acid sequence of polypeptide, promptly CSRRGELAASRRGELC(SEQ ID NO:17) design.These new compounds are referred to as hB in the text _AG2, riB _AG1And hriB _AG2, be listed in the table below 1.

Table I: Trk receptor agonist compounds

Identifier	Sequence	Key word
			hB AG2	c(SRRGELSRRGEL)(SEQ ID NO：39)	The peptide bond of cyclisation
riB AG1	Ac-dCdLdEGdRdRdSdAdAdLdEGdRdRdSdC-NIIs (SEQ ID NO：40)	The D-amino acid residue is by the cyclisation of cysteine disulfide bond
			hriB AG2	c(dLdEGdRdRdSdLdEGdRdRdS)(SEQ ID NO：41)	The peptide bond of D-amino acid residue cyclisation

In above-mentioned Table I, lower case " c " is used to represent by peptide or amido link the amino terminal amino acid residue is connected to the cyclisation that carboxyamino acid residue forms.Therefore, hB _AG1Polypeptide (SEQID NO:39) preferably comprises the amido link that the terminal serine residue of N-is connected to the terminal leucine residue of C-.Similarly, peptide hriB _AG2(SEQ ID NO:41) preferably comprises the amido link that the terminal leucine residue of N-is connected to the C-terminal serine residue.

Lower case " d " in the Table I before the amino acid residue is used to represent that this residue is D-amino acid residue (relative with the L-amino acid residue).Therefore, polypeptide riB _AG1And hriB _AG2Preferably comprise the D-amino acid residue.In fact, the amino acid residue in these polypeptide (except the glycine residue, it is neither L neither the D amino acid residue) all is preferably the D-amino acid residue.

Obviously, through visual observation riB _AG1And hriB _AG2Aminoacid sequence (SEQ ID NOS:40 and 41), these sequences are B _AGThe reverse sequence of peptide sequence (SEQ ID NO:17).Especially, as be understood by one of ordinary skill in the art, the expection of the polypeptide of the D-of containing amino acid residue of the present invention will be taked the three dimensional structure (i.e. " conformation ") similar or identical basically with the three dimensional structure that contains L-aminoacid reverse sequence polypeptide.Therefore, except the polypeptide of above-mentioned L-amino acid residue, focus attentions equally on of the present invention has the polypeptide of D-amino acid residue reverse sequence.Therefore, in a preferred embodiment, peptide of the present invention and peptide mimics comprise the sequence of the L-amino acid residue that has comprised above-mentioned Arg-Gly-Glu (i.e. " RGE ") motif.Correspondingly, the present invention also provides such peptide and peptide mimics in an alternate embodiment, and it comprises the sequence of the D-amino acid residue that has comprised short-term sequence motifs dGlu-Gly-dArg (i.e. " dEGdR ").

Comprise for example Proteolytic enzyme enzymatic degradation of the more stable in vivo and difficult quilt of the peptide of the present invention of this D-amino acid residue and peptide mimics expection.Similarly, the cyclic amides key for example is used in riB _AG1And hriB _AG2In the polypeptide those, same expection is difficult for degradation in vivo.The peptide that shortens is (as hB _AG, itself and B _AGCompare disappearance two terminal cysteine and two central alanine) more likely pass through blood brain barrier.Therefore, this peptide can for example, be used for pharmaceutical composition and individual administration by preferably.

7.8.2 biological activity

In test, tested hB based on substrate _AG2, riB _AG1And hriB _AG2Polypeptide promotes the ability that neurite grows to estimate it in suppressing environment.Especially, as discussed above such, myelin associated glycoprotein (MAG) before had been proved to be the inhibition neurite and had grown reaction.Referring to, for example, people such as Mukhopadhyay, " neuron " (Neuron) 1994,13:757-767; And people such as Tang, " molecular cell neuroscience " (M0l.Cll.NeurosCi.) 1997,9:333-346.Shown in above-mentioned embodiment, Trk receptor stimulating agent such as B _AGPolypeptide can seal MAG and suppress active, and promotes that neurite suppresses to grow in the environment (promptly existing under the situation of MAG) at this.The data that these experiments of describing in the literary composition present show hB _AG2, riB _AG1And hriB _AG2Polypeptide seals MAG equally to be suppressed active and promotes neurite to grow.

Material and method

Briefly, standard plastic 8 Room tissue culture slides are coated as follows: (a) polylysine; (b) mixture of polylysine and goat-anti-human IgG and fibronectin; The perhaps mixture of (c) polylysine, goat-anti-human IgG (Fc-is specific) and fibronectin and MAG-Fc.At first, at room temperature with being dissolved in distilled water (" dH ₂O ") 17 μ g/ml polylysine bags by microscope slide 30 (30) minutes.After blot in the hole, will resist the mixture of human IgG and/or fibronectin (both is dissolved among the DMEM, 10 μ g/ml) to add in the hole of stand-by these chemical compound bag quilts, hatched 120 minutes.Blot once more in the hole, and (for the Kong Eryan with MAG-Fc bag quilt) hatched 60 (60) minutes with MAG-Fc (0.25 μ g/ml is dissolved in DMEM and 10%FCS).Be dissolved in DMEM to each hole with the 15K adding afterwards, 10%FCS, the PND2/3 rat cerebellum neuron of 25mM KCl and 5ng/ml FGF2 makes the culture medium final volume in each hole reach 300 μ l.Before with fixing GAP-43 and dyeing, cultivated cerebellar neuron 72 hours.Polylysine, goat-anti-human IgG (Fc-is specific) and fibronectin available from SIGMA (St.Louis, Missouri).

The result.Measured each neuron average length of long neurite.Observed background neurite grows about 9 μ m on the polylysine substrate.Be increased to about 24 μ m prominent the growing of polylysine/fibronectin substrate epineural.Neurite grows and drops to about 15 μ m in the hole that extra MAG-Fc coating is arranged.Figure 18 A has shown the dose-effect curve of three kinds of peptide mimicses.Peptide mimics hriB _AG2(SEQ ID NO:40) promoted to suppress the great dose dependent growth of neurite under the environment.Under the dosage of about 10 μ g/ml, can be observed the growth response of neurite, and under the dosage of 33 μ g/ml (maximum concentration of measurement), almost be the twice of visible value in not having the inhibition environment of peptide mimics.HB _AG2(SEQ ID NO:39) promotes the growth of neurite under the dosage of 33 μ g/ml.RiB _AGl(SEQ ID NO:41) do not promote growth under same concentration.

Figure 18 B has shown and has described to have BDNF, B _AG, hriB _AG2, hB _AG2Or riB _AGThe inhibition environment in the bar chart of neurite outgrowth.BDNF does not have effect to neurite outgrowth under the concentration of 10 μ g/ml and 100 μ g/ml.B _AGPeptide promotes neurite outgrowth under the concentration of 10 μ g/ml and 100 μ g/ml.HriB _AG2Promote neurite outgrowth under the concentration of 33 μ g/ml, its degree is far above BDNF, B _AGHB under peptide and any concentration _AG2And riB _AG1HB _AG2Under the concentration of 33 μ g/ml, promote neurite outgrowth, the B under the concentration of its degree and 10 μ g/ml _AGPolypeptide is suitable.

These results show other peptide and peptide mimics, for example the application B _AGThe derivant of peptide can promote neurite outgrowth and reaches and B in suppressing environment _AGPolypeptide suitable or even better degree.

7.9.p75 receptor-binding agents is restrained the inhibition of neure growth

The experimentation that present embodiment is described the effect of p75 receptor-binding agents under the environment that suppresses neure growth usually.Especially, experimental results show that the p75 receptor-binding agents has resisted the inhibition activity that suppresses molecule and/or its receptor.

Neurite grows test

With people such as previous Williams (" neuron " (Neuron) 1994,13:583-594) described method is at monolayer parent 3T3 cell or LK8 cell (the transfection 3T3cells of the chicken N-cadherin of the expressed physiological level of foundation; Referring to people such as Doherty, " neuron " (Neuron) 1991 set up cerebellar neuron on 6:247-258) and cultivated altogether.Cell maintains in the DulbeccoShi improvement EagleShi culture medium of having replenished 10% hyclone (FCS).These cerebellar neurons are expressed the TrkB receptor but the TrkA receptor of non-expressive function level.

For setting up coculture, with about 80,000 3T3 cells (or LK8 cell) coated plate in each chamber of the tissue culture's slide that is coated with poly-L-Lysine and fibronectin with eight Room.The coated plate cell spends the night and maintains in the DulbeccoShi improvement EagleShi culture medium of having replenished 10%FCS, to form confluent monolayer.Remove culture medium and will about 6,000 dissociated cerebellar neurons (taking from the back 2/3 day rat that is born) coated plate in each hole that has replenished 2%FCS SATO culture medium.Adding test agent as described herein is also kept common cultivation 23 hours.Coculture is fixed and the dyeing of GAP-43 immunoreactivity then.Neuron between about 120 to 150 is measured the average length of the longest neurite of each cell, equally as people such as Williams (" neuron " (Neuron) 1994,13:583-594) before described.

Reagent

Recombined human NGF and BDNF are available from R﹠amp; D system (Minneapolis, MN).Synthetic peptide all available from commercial supplier (Multiple Peptide Systems, San Diego, CA).All peptides use conventional method with reversed phase high-performance liquid chromatography (RP-HPLC) purification, and obtain (as being higher than 95% purity) with the highest purity level.

The p75 receptor-binding agents promotes neurotrophin-mediated neure growth in suppressing environment.Before proved when myelin associated glycoprotein (MAG) as cytostromatic transfection molecule (people such as Mukhopadhyay, " neuron " (Neuron) 1994,13:757-767) present or as solubility Fc chimeric protein (people such as Tang, " molecular cell neuroscience " (Mol.Cell.Neurosci.) 1997 9:333-346) adds and can suppress neurite when giving birth back 2-3 days rat cerebellum neuron and grow reaction.In order to go deep into those research, the back 3 days cerebellar neuron of birth is incubated on the monolayer LK8 cell, this cell is a kind of 3T3 fibroblast that can express transfection N-cadherin, and formerly proved the powerful growth response (people such as Williams that can promote neurite, " neuron " (Neuron) 1994,13:583-594).Cell is incubated at there is concentration be in the warm proteic culture medium of 0,5 or 25 μ g/ml soluble M AG-Fc.Just as was expected, and MAG-Fc has suppressed the growth of neurite in the mode of dose dependent, and when the concentration that exists in the culture medium was 25 μ g/ml, inhibitory reaction was about 40%.Therefore, containing warm proteic this culture medium of soluble M AG-Fc is the inhibition culture medium.

It is bonded that this inhibition culture medium has further been replenished NGF, BDNF (1ng/ml) the associating NGF (10ng/ml or 100ng/ml) of BDNF, the 10ng/ml of 1ng/ml or 100ng/ml, and 100 μ g/ml encircle 1 motif (N-Ac-in conjunction with the NGF of p75 receptor CTDIKGKEC-NH ₂) constraint monomer or NGF encircle 1 peptide (100 μ g/ml) Associated with BDNF (1ng/ml).The growth medium that only contains MAG-Fc in contrast.When neurotrophin BDNF is present in the inhibition culture medium with the concentration of 1ng/ml not at the MAG reaction the effect surveyed, just, MAG-Fc continues to suppress neurite and grows.The same with BDNF, NGF does not stimulate the neurite under the MAG-Fc existence to grow (Figure 19) under the concentration of 10ng/ml or 100ng/ml.It seems from individual experimental data, use the result that NGF obtained of 10ng/ml and 100ng/ml not have tangible difference, and therefore these data are merged.These results conform to previous report, promptly neuronal cell must with neurotrophin sensitization with stop MAG and myelinic inhibition activity (referring to people such as Cai, " neuron " (Neuron) 1999,22:89-101).

Independent NGF encircles 1 binding motif constraint monomer and when having MAG-Fc neurite is grown and also do not have effect (Figure 19).Yet the NGF of Associated with BDNF encircles 1 peptide and has but produced tangible neurite and grow reaction.In addition, when BDNF and NGF are added (Figure 19) simultaneously with the ratio of 1: 10 or 1: 100 (BDNF is than NGF), also observe tangible neurite and grow reaction.

These experimental results show that when administration BDNF and NGF in the inhibition environment, NGF allows BDNF to promote that neurite grows.This result shows that also administration NGF first beta hairpin ring constraint monomer allows BDNF to promote to suppress the neuronic growth of CNS in the environment.

7.10. the patient of treatment spinal cord injury

A patient who is diagnosed as the breast spinal cord injury has lost the consciousness and the motor capacity of shank.This patient has been carried out operation with the fixing spine of chest.After to soft tissue and bone debridement, expose impaired spinal cord.The sterilization medicated powder that contains the p75 receptor-binding agents mixes with the disinfectant ordinary salt to form gelinite.The surgeon uses p75 receptor-binding agents gel at the spinal cord surface local that exposes.Finish fixing the processing with usual way.Postoperative has been monitored the consciousness of patient's lower limb and/or the improvement of motor capacity.

8. the list of references of quoting

In description of the present invention, quote and discussed a large amount of lists of references, comprise patent, patent application and various publication.Quoting and/or discussing of these lists of references only supplies to be used for illustrating explanation of the present invention, and is not to admit that arbitrary reference material is " prior art " of the present invention described herein.All lists of references that this description is quoted and/or discussed (comprising quoting biological sequence in for example GenBank, PDB or other public database or structure) are whole to be incorporated herein by reference at this, just is incorporated herein by reference separately as every piece of list of references.

Claims (105)

1. a cyclic peptide comprises aminoacid sequence: Arg-Gly-Glu in the ring ring of described cyclic peptide,

Wherein, this cyclic peptide is regulated the receptor-mediated activity of Trk.

2. according to the cyclic peptide of claim 1, wherein the receptor-mediated activity of Trk is selected from: neure growth, neuronal survival, axon growth, synaptic plasticity and neurite grow.

3. according to the cyclic peptide of claim 1, it is regulated neurite and grows.

4. according to the cyclic peptide of claim 1, it suppresses the activity of Trk mediation.

5. according to the cyclic peptide of claim 1, it strengthens the activity of Trk mediation.

6. according to the cyclic peptide of claim 1, this cyclic peptide comprises structural formula:

Wherein:

(a) Y ₁And Y ₂Be independently selected from and have the Y of being formed on ₁And Y ₂Between the aminoacid of covalent bond; With

(b) X ₁And X ₂Choose wantonly, and if present, the aminoacid sequence that is independently selected from aminoacid or connects by peptide bond.

7. according to the cyclic peptide of claim 6, wherein the magnitude range of cyclic peptide ring is from 5 to 15 aminoacid.

8. according to the cyclic peptide of claim 6, described cyclic peptide has structural formula:

，

, or

。

9. cyclic peptide according to Claim 8, described cyclic peptide has aminoacid sequence:

Cys-Arg-Gly-Glu-Cys(SEQ ID NO：9)；

Cys-Ser-Arg-Arg-Gly-Glu-Cys(SEQ ID NO：1)；

Cys-Ala-Arg-Arg-Gly-Glu-Cys(SEQ ID NO：3)；

Cys-Phe-His-Arg-Gly-Glu-Cys (SEQID NO:5); Or

Cys-Ser-His-Arg-Gly-Glu-Cys(SEQ ID NO：7)；

Wherein, a covalent bond connects the terminal and C-terminal cysteine of N-in the described aminoacid sequence.

10. according to the cyclic peptide of claim 6, it suppresses the activity of Trk mediation.

11. according to the cyclic peptide of claim 6, this cyclic peptide comprises structural formula:

，

Wherein:

(a) Y ₁And Y ₂Be independently selected from and have the Y of being formed on ₁And Y ₂Between the aminoacid of covalent bond; With

(b) Z ₁, Z ₂And Z ₀Choose wantonly, and if present, the aminoacid sequence that is independently selected from aminoacid or connects by peptide bond.

12. according to the cyclic peptide of claim 11, wherein the magnitude range of cyclic peptide ring is about 8-50 amino acid residue.

13. according to the cyclic peptide of claim 11, described cyclic peptide has structural formula:

。

14. according to the cyclic peptide of claim 11, described cyclic peptide has aminoacid sequence: Cys-Ser-Arg-Arg-Gly-Glu-Leu-Ala-Ala-Ser-Arg-Arg-Gly-Glu-Leu-Cys (SEQ ID NO:17),

Wherein, a covalent bond connects the terminal and C-terminal cysteine of N-in the described aminoacid sequence.

15. according to the cyclic peptide of claim 11, it strengthens the activity of Trk mediation.

16. according to claim 1,6,8-9,11 and each cyclic peptide of 13-14, described cyclic peptide also comprises N-acetyl group, N-formoxyl or N-mesyl on n terminal residue.

17. according to claim 1,6,8-9,11,13-14 and 16 each cyclic peptide, described cyclic peptide also comprises the C-amide groups on the C-terminal residue.

18. according to claim 6,8,11 and 13 each cyclic peptide, wherein Y ₁And Y ₂Covalently bound by disulfide bond.

19. according to the cyclic peptide of claim 18, wherein Y ₁And Y ₂Independently be selected from penicillamine, β, β-tetramethylene cysteine, β, β-pentylidene cysteine, β-Qiu Jibingsuan, β, β-pentylidene β-Qiu Jibingsuan, 2-sulfydryl benzene, 2-mercaptoaniline, 2-sulfydryl proline and their derivant.

20. according to the cyclic peptide of claim 19, wherein Y ₁And Y ₂It is respectively the cysteine or derivatives thereof.

21. according to claim 6,8,11 and 13 each cyclic peptide, wherein Y ₁And Y ₂Covalently bound by amido link.

22. according to the cyclic peptide of claim 21, wherein amido link is formed between the functional end-group.

23. according to the cyclic peptide of claim 21, wherein amido link is formed between a functional end-group and the residue side chain.

24. according to the cyclic peptide of claim 22, wherein:

(a) Y ₁Be selected from lysine, ornithine and their derivant; With

(b) Y ₂Be selected from aspartic acid, glutamic acid and their derivant.

25. according to the cyclic peptide of claim 22, wherein:

(a) Y ₁Be selected from aspartic acid, glutamic acid and their derivant; With

(b) Y ₂Be selected from lysine, ornithine and their derivant.

26. according to claim 6,8,11 and 13 each cyclic peptide, wherein Y ₁And Y ₂Covalently bound by thioether bond.

27. according to claim 6,8,11 and 13 each cyclic peptide, wherein:

(a) Y ₁And Y ₂It is respectively the tryptophan or derivatives thereof; With

(b) Y ₁And Y ₂Between covalent bond form δ ₁δ ₁-.delta.1.delta.1'-Ditryptophan. or derivatives thereof.

28. one kind is screened the method that candidate compound is regulated the receptor-mediated active ability of Trk, this method comprises that the three dimensional structure with candidate compound compares with the three dimensional structure of regulating the receptor-mediated active cyclic peptide of Trk, wherein:

(a) described cyclic peptide in its ring ring, comprise aminoacid sequence Arg-Gly-Glu and

(b) similarity between candidate compound structure and the cyclic peptide structures shows the receptor-mediated active ability of this candidate compound adjusting Trk.

29. according to the method for claim 28, wherein cyclic peptide comprises structural formula:

，

(a) Y ₁And Y ₂Be independently selected from and have the Y of being formed on ₁And Y ₂Between the aminoacid of covalent bond; With

(b) X ₁And X ₂Choose wantonly, and if present, the aminoacid sequence that is independently selected from aminoacid or connects by peptide bond.

30. according to the method for claim 28, wherein cyclic peptide comprises structural formula:

，

Wherein:

(a) Y ₁And Y ₂Be independently selected from and have the Y of being formed on ₁And Y ₂Between the aminoacid of covalent bond; With

(b) Z ₁, Z ₂And Z ₀Choose wantonly, and if present, the aminoacid sequence that is independently selected from aminoacid or connects by peptide bond.

31. according to the method for claim 28, wherein cyclic peptide comprises and is selected from following aminoacid sequence:

Cys-Arg-Gly-Glu-Cys(SEQ ID NO：9)；

Cys-Ser-Arg-Arg-Gly-Glu-Cys(SEQ ID NO：1)；

Cys-Ala-Arg-Arg-Gly-Glu-Cys(SEQ ID NO：3)；

Cys-Phe-His-Arg-Gly-Glu-Cys(SEQID NO：5)；

Cys-Ser-His-Arg-Gly-Glu-Cys (SEQ ID NO:7); With

Cys-Ser-Arg-Arg-Gly-Glu-Leu-Ala-Ala-Ser-Arg-Arg-Gly-Glu-Leu-Cys(SEQ ID NO：17)；

Wherein, a covalent bond connects the terminal and C-terminal cysteine of N-in the described aminoacid sequence.

32. according to the method for claim 28, wherein:

(a) this cyclic peptide strengthen the receptor-mediated activity of Trk and

(b) similarity between candidate compound structure and the cyclic peptide structures shows that this candidate compound strengthens the receptor-mediated active ability of Trk.

33. according to the method for claim 28, wherein:

(a) this cyclic peptide suppress the receptor-mediated activity of Trk and

(b) similarity between candidate compound structure and the cyclic peptide structures shows that this candidate compound suppresses the receptor-mediated active ability of Trk.

34. regulate the receptor-mediated active peptide mimics of Trk for one kind, wherein this peptide mimics has the similar basically three dimensional structure of three dimensional structure of the active cyclic peptide that mediates to adjusting Trk, described cyclic peptide comprises aminoacid sequence Arg-Gly-Glu in its ring.

35. according to the peptide mimics of claim 34, wherein cyclic peptide comprises structural formula:

，

Wherein:

(a) Y ₁And Y ₂Be independently selected from and have the Y of being formed on ₁And Y ₂Between the aminoacid of covalent bond; With

(b) X ₁And X ₂Choose wantonly, and if present, the aminoacid sequence that is independently selected from aminoacid or connects by peptide bond.

36. according to the peptide mimics of claim 34, wherein cyclic peptide comprises structural formula:

，

Wherein:

(a) Y ₁And Y ₂Be independently selected from and have the Y of being formed on ₁And Y ₂Between the aminoacid of covalent bond; With

(b) Z ₁, Z ₂And Z ₀Choose wantonly, and if present, the aminoacid sequence that is independently selected from aminoacid or connects by peptide bond.

37. according to the peptide mimics of claim 34, wherein cyclic peptide comprises and is selected from following aminoacid sequence:

Cys-Arg-Gly-Glu-Cys(SEQ ID NO：9)；

Cys-Ser-Arg-Arg-Gly-Glu-Cys(SEQ ID NO：1)；

Cys-Ala-Arg-Arg-Gly-Glu-Cys(SEQ ID NO：3)；

Cys-Phe-His-Arg-Gly-Glu-Cys(SEQID NO：5)；

Cys-Ser-His-Arg-Gly-Glu-Cys (SEQ ID NO:7); With

Cys-Ser-Arg-Arg-Gly-Glu-Leu-Ala-Ala-Ser-Arg-Arg-Gly-Glu-Leu-Cys(SEQ ID NO：17)；

Wherein, a covalent bond connects the terminal and C-terminal cysteine of N-in the described aminoacid sequence.

38. according to the peptide mimics of claim 34, it strengthens the receptor-mediated activity of Trk.

39. according to the peptide mimics of claim 34, it suppresses the receptor-mediated activity of Trk.

40. one kind is suppressed the receptor-mediated active method of Trk, this method comprise with cell with a certain amount of according to claim 4 cyclic peptide or contact according to the peptide mimics of claim 39, wherein effectively suppress the receptor-mediated activity of Trk with the cyclic peptide of cells contacting or the amount of peptide mimics.

41. according to the method for claim 40, wherein the receptor-mediated activity of Trk is selected from: neure growth, neuronal survival, axon growth, synaptic plasticity and neurite grow.

42. according to the method for claim 40, wherein cell contacts with the cyclic peptide with following structural formula:

，

Wherein:

(a) Y ₁And Y ₂Be independently selected from and have the Y of being formed on ₁And Y ₂Between the aminoacid of covalent bond; With

(b) X ₁And X ₂Choose wantonly, and if present, the aminoacid sequence that is independently selected from aminoacid or connects by peptide bond.

43. according to the method for claim 42, wherein cell contacts with the cyclic peptide with following structural formula:

，

，

，

, or

44. according to the method for claim 43, wherein cell contacts with the cyclic peptide with following aminoacid sequence:

Cys-Arg-Gly-Glu-Cys(SEQ ID NO：9)；

Cys-Ser-Arg-Arg-Gly-Glu-Cys(SEQ ID NO：1)；

Cys-Ala-Arg-Arg-Gly-Glu-Cys(SEQ ID NO：3)；

Cys-Phe-His-Arg-Gly-Glu-Cys (SEQID NO:5); Or

Cys-Ser-His-Arg-Gly-Glu-Cys(SEQ ID NO：7)；

Wherein, a covalent bond connects the terminal and C-terminal cysteine of N-in the described aminoacid sequence.

45. according to the method for claim 40, wherein cell contacts with peptide mimics, the three dimensional structure that described peptide mimics has is similar basically to the three dimensional structure of the cyclic peptide with following structural formula:

，

Wherein:

(a) Y ₁And Y ₂Be independently selected from and have the Y of being formed on ₁And Y ₂Between the aminoacid of covalent bond; With

(b) X ₁And X ₂Choose wantonly, and if present, the aminoacid sequence that is independently selected from aminoacid or connects by peptide bond.

46. according to the method for claim 45, wherein the three dimensional structure of peptide mimics three dimensional structure that has and the cyclic peptide with following structural formula is similar basically:

，

，

，

, or

。

47. according to the method for claim 46, wherein the three dimensional structure of peptide mimics three dimensional structure that has and the cyclic peptide with following aminoacid sequence is similar basically:

Cys-Arg-Gly-Glu-Cys(SEQ ID NO：9)；

Cys-Ser-Arg-Arg-Gly-Glu-Cys(SEQ ID NO：1)；

Cys-Ala-Arg-Arg-Gly-Glu-Cys(SEQ ID NO：3)；

Cys-Phe-His-Arg-Gly-Glu-Cys (SEQID NO:5); Or

Cys-Ser-His-Arg-Gly-Glu-Cys(SEQ ID NO：7)；

Wherein, a covalent bond connects the terminal and C-terminal cysteine of N-in the described aminoacid sequence.

48. one kind strengthens the receptor-mediated active method of Trk, this method comprise with cell with a certain amount of according to claim 5 cyclic peptide or contact according to the peptide mimics of claim 38, wherein effectively suppress the receptor-mediated activity of Trk with the cyclic peptide of cells contacting or the amount of peptide mimics.

49. according to the method for claim 48, wherein the receptor-mediated activity of Trk is selected from: neure growth, neuronal survival, axon growth, synaptic plasticity and neurite grow.

50. according to the method for claim 48, wherein cell contacts with the cyclic peptide with following structural formula:

，

Wherein:

(a) Y ₁And Y ₂Be independently selected from and have the Y of being formed on ₁And Y ₂Between the aminoacid of covalent bond; With

(b) Z ₁, Z ₂And Z ₀Choose wantonly, and if present, the aminoacid sequence that is independently selected from aminoacid or connects by peptide bond.

51. according to the method for claim 50, wherein cyclic peptide has structural formula:

。

52. according to the method for claim 51, wherein cyclic peptide comprises aminoacid sequence:

Cys-Ser-Arg-Arg-Gly-Glu-Leu-Ala-Ala-Ser-Arg-Arg-Gly-Glu-Leu-Cys(SEQ ID NO：17)，

Wherein, a covalent bond connects the terminal and C-terminal cysteine of N-in the described aminoacid sequence.

53. according to the method for claim 48, wherein cell contacts with peptide mimics, the three dimensional structure that described peptide mimics has is similar basically to the three dimensional structure of the cyclic peptide with following structural formula:

，

Wherein:

(a) Y ₁And Y ₂Be independently selected from and have the Y of being formed on ₁And Y ₂Between the aminoacid of covalent bond; With

(b) Z ₁, Z ₂And Z ₀Choose wantonly, and if present, the aminoacid sequence that is independently selected from aminoacid or connects by peptide bond.

54. according to the method for claim 53, wherein the three dimensional structure of peptide mimics three dimensional structure that has and the cyclic peptide with following structural formula is similar basically:

。

55. according to the method for claim 54, wherein the three dimensional structure of peptide mimics three dimensional structure that has and the cyclic peptide that comprises following aminoacid sequence is similar basically:

Cys-Ser-Arg-Arg-Gly-Glu-Leu-Ala-Ala-Ser-Arg-Arg-Gly-Glu-Leu-Cys(SEQ ID NO：17)，

Wherein, a covalent bond connects the terminal and C-terminal cysteine of N-in the described aminoacid sequence.

56. one kind promotes individual central nervous system (CNS) growth or the method for repairing, this method comprise give individual a certain amount of according to claim 5 cyclic peptide or according to the peptide mimics of claim 38, the cyclic peptide that wherein gives or the amount of peptide mimics effectively promote the CNS growth or repair.

57. according to the method for claim 56, wherein to individual administration cyclic peptide, this cyclic peptide has following structural formula:

，

Wherein:

(a) Y ₁And Y ₂Be independently selected from and have the Y of being formed on ₁And Y ₂Between the aminoacid of covalent bond; With

(b) Z ₁, Z ₂And Z ₀Choose wantonly, and if present, the aminoacid sequence that is independently selected from aminoacid or connects by peptide bond.

58. according to the method for claim 57, wherein cyclic peptide has structural formula:

。

59. according to the method for claim 58, wherein cyclic peptide comprises aminoacid sequence:

Cys-Ser-Arg-Arg-Gly-Glu-Leu-Ala-Ala-Ser-Arg-Arg-Gly-Glu-Leu-Cys(SEQ ID NO：17)，

Wherein, a covalent bond connects the terminal and C-terminal cysteine of N-in the described aminoacid sequence.

60. according to the method for claim 56, wherein to individual administration peptide mimics, the three dimensional structure that this peptide mimics has is similar basically to the three dimensional structure of the cyclic peptide with following structural formula:

，

Wherein:

(a) Y ₁And Y ₂Be independently selected from and have the Y of being formed on ₁And Y ₂Between the aminoacid of covalent bond; With

(b) Z ₁, Z ₂And Z ₀Choose wantonly, and if present, the aminoacid sequence that is independently selected from aminoacid or connects by peptide bond.

61. according to the method for claim 60, wherein the three dimensional structure of peptide mimics three dimensional structure that has and the cyclic peptide with following structural formula is similar basically:

。

62. according to the method for claim 61, wherein the three dimensional structure of peptide mimics three dimensional structure that has and the cyclic peptide that comprises following aminoacid sequence is similar basically:

Cys-Ser-Arg-Arg-Gly-Glu-Leu-Ala-Ala-Ser-Arg-Arg-Gly-Glu-Leu-Cys(SEQ ID NO：17)，

Wherein, a covalent bond connects the terminal and C-terminal cysteine of N-in the described aminoacid sequence.

63. a pharmaceutical composition, it comprises:

(a) a certain amount of according to claim 1 cyclic peptide or according to the peptide mimics of claim 34, described amount can effectively be regulated the receptor-mediated activity of Trk; With

(b) one or more pharmaceutically or the physiology go up acceptable carrier, diluent or excipient.

64. according to the pharmaceutical composition of claim 63, wherein cyclic peptide or peptide mimics strengthen the receptor-mediated activity of Trk.

65. according to the pharmaceutical composition of claim 63, wherein cyclic peptide or peptide mimics suppress the receptor-mediated activity of Trk.

66. method that reduces the reaction of CNS inhibitor, this method comprise with cell with a certain amount of according to claim 5 cyclic peptide or contact according to the peptide mimics of claim 38, wherein can effectively reduce the reaction of CNS inhibitor with the cyclic peptide of cells contacting or the amount of peptide mimics.

67. according to the method for claim 66, wherein the reaction of CNS inhibitor is by the signal cascade mediation, described signal cascade has one or more components that regulated by protein kinase A (PKA) or phosphoinositide-3 kinases (P13K).

68. according to the method for claim 66, wherein the reaction of CNS inhibitor is by relating to the signal cascade mediation of Rho.

69. according to the method for claim 66, wherein the reaction of CNS inhibitor is by MAG, Nogo-A, oligodendroglia myelin glycoprotein, NgR, GT1b, p75 ^NTR, the mediation of chondroitin sulfate proteoglycan or semaphorins reaction.

70. according to the method for claim 66, wherein cell contacts with the cyclic peptide with following structural formula:

，

Wherein:

(a) Y ₁And Y ₂Be independently selected from and have the Y of being formed on ₁And Y ₂Between the aminoacid of covalent bond; With

(b) Z ₁, Z ₂And Z ₀Choose wantonly, and if present, the aminoacid sequence that is independently selected from aminoacid or connects by peptide bond.

71. according to the method for claim 70, wherein cyclic peptide has structural formula:

。

72. according to the method for claim 71, wherein cyclic peptide comprises aminoacid sequence:

Cys-Ser-Arg-Arg-Gly-Glu-Leu-Ala-Ala-Ser-Arg-Arg-Gly-Glu-Leu-Cys(SEQ ID NO：17)，

Wherein, a covalent bond connects the terminal and C-terminal cysteine of N-in the described aminoacid sequence.

73. according to the method for claim 66, wherein cell contacts with peptide mimics, the three dimensional structure that described peptide mimics has is similar basically to the three dimensional structure of the cyclic peptide with following structural formula:

，

Wherein:

(a) Y ₁And Y ₂Be independently selected from and have the Y of being formed on ₁And Y ₂Between the aminoacid of covalent bond; With

(b) Z ₁, Z ₂And Z ₀Choose wantonly, and if present, the aminoacid sequence that is independently selected from aminoacid or connects by peptide bond.

74. according to the cyclic peptide of claim 73, wherein the three dimensional structure of peptide mimics three dimensional structure that has and the cyclic peptide with following structural formula is similar basically:

。

75. according to the method for claim 74, wherein the three dimensional structure of peptide mimics three dimensional structure that has and the cyclic peptide that comprises following aminoacid sequence is similar basically:

Cys-Ser-Arg-Arg-Gly-Glu-Leu-Ala-Ala-Ser-Arg-Arg-Gly-Glu-Leu-Cys(SEQ ID NO：17)，

Wherein, a covalent bond connects the terminal and C-terminal cysteine of N-in the described aminoacid sequence.

76. a method that reduces the reaction of individual CNS inhibitor, this method comprise give individual a certain amount of according to claim 5 cyclic peptide or according to the peptide mimics of claim 38, the cyclic peptide that wherein gives or the amount of peptide mimics effectively reduce the reaction of CNS inhibitor.

77. according to the method for claim 76, wherein the reaction of CNS inhibitor is by the signal cascade mediation, described signal cascade has one or more components that regulated by protein kinase A (PKA) or phosphoinositide-3 kinases (P13K).

78. according to the method for claim 76, wherein the reaction of CNS inhibitor is by relating to the signal cascade mediation of Rho.

79. according to the method for claim 76, wherein the reaction of CNS inhibitor is by MAG, Nogo-A, oligodendroglia myelin glycoprotein, NgR, GT1b, p75 ^NTR, the mediation of chondroitin sulfate proteoglycan or semaphorins reaction.

80. according to the method for claim 76, wherein cell contacts with the cyclic peptide with following structural formula:

，

Wherein:

(a) Y ₁And Y ₂Be independently selected from and have the Y of being formed on ₁And Y ₂Between the aminoacid of covalent bond; With

(b) Z ₁, Z ₂And Z ₀Choose wantonly, and if present, the aminoacid sequence that is independently selected from aminoacid or connects by peptide bond.

81. 0 method according to Claim 8, wherein cyclic peptide has structural formula:

。

82. 1 method according to Claim 8, wherein cyclic peptide comprises aminoacid sequence:

Cys-Ser-Arg-Arg-Gly-Glu-Leu-Ala-Ala-Ser-Arg-Arg-Gly-Glu-Leu-Cys(SEQ ID NO：17)，

Wherein, a covalent bond connects the terminal and C-terminal cysteine of N-in the described aminoacid sequence.

83. according to the method for claim 76, wherein cell contacts with peptide mimics, the three dimensional structure that described peptide mimics has is similar basically to the three dimensional structure of the cyclic peptide with following structural formula:

，

Wherein:

(a) Y ₁And Y ₂Be independently selected from and have the Y of being formed on ₁And Y ₂Between the aminoacid of covalent bond; With

(b) Z ₁, Z ₂And Z ₀Choose wantonly, and if present, the aminoacid sequence that is independently selected from aminoacid or connects by peptide bond.

84. 3 method according to Claim 8, wherein the three dimensional structure of peptide mimics three dimensional structure that has and the cyclic peptide with following structural formula is similar basically:

。

85. 4 method according to Claim 8, wherein the three dimensional structure of peptide mimics three dimensional structure that has and the cyclic peptide that comprises following aminoacid sequence is similar basically:

Cys-Ser-Arg-Arg-Gly-Glu-Leu-Ala-Ala-Ser-Arg-Arg-Gly-Glu-Leu-Cys(SEQ ID NO：17)，

Wherein, a covalent bond connects the terminal and C-terminal cysteine of N-in the described aminoacid sequence.

86. a cyclic peptide comprises the D aminoacid sequence in the ring ring of described cyclic peptide:

dGlu-Gly-dArg，

Wherein, this cyclic peptide is regulated the receptor-mediated activity of Trk.

87. 6 cyclic peptide according to Claim 8, it promotes neurite outgrowth.

88. 6 cyclic peptide according to Claim 8, this cyclic peptide comprises structural formula:

，

Wherein:

(a) Y ₁And Y ₂Be independently selected from and have the Y of being formed on ₁And Y ₂Between the aminoacid of covalent bond; With

(b) X ₁And X ₂Choose wantonly, and if present, the aminoacid sequence that is independently selected from aminoacid or connects by peptide bond.

89. 8 cyclic peptide wherein is formed on Y according to Claim 8 ₁And Y ₂Between covalent bond be peptide bond.

90. 9 cyclic peptide according to Claim 8, described cyclic peptide has aminoacid sequence:

dLeu-dGlu-Gly-dArg-dArg-dSer-dLeu-dGlu-Gly-dArg-dArg-dSer(SEQ ID NO：40)。

91. 6 cyclic peptide according to Claim 8, it comprises the structural formula with following aminoacid sequence:

dCys-dLeu-dGlu-Gly-dArg-dArg-dSer-dAla-dAla-dLeu-dGlu-Gly-dArg-dArg-dSer-dCys(SEQ ID NO：41)；

Wherein, terminal cysteine is covalently bound by disulfide bond.

92. cyclic peptide according to Claim 8, described cyclic peptide has aminoacid sequence:

Ser-Arg-Arg-Gly-Glu-Leu-Ser-Arg-Arg-Gly-Glu-Leu(SEQ ID NO：39)；

Wherein, terminal serine and terminal leucine are covalently bound by peptide bond.

93. a method that promotes to suppress the CNS neure growth in the environment, this method comprises the p75 receptor-binding agents that gives the individual treatment effective dose.

94. according to the method for claim 93, wherein the p75 receptor-binding agents comprises neurotrophin binding motif or its peptide mimics.

95. according to the method for claim 94, wherein the p75 receptor-binding agents is cyclic peptide or the peptide mimics that comprises following aminoacid sequence in its ring ring:

Thr-Asp-Ile-Lys-Gly-Lys-Glu(TDIKGKE)(SEQ ID NO：42)。

96. according to the method for claim 95, wherein the p75 receptor-binding agents is N-Ac- CTDIKGKEC-NH ₂(SEQ ID NO:43).

97. according to the method for claim 93, it also comprises and gives neurotrophin.

98. according to the method for claim 97, wherein the p75 receptor-binding agents is to arrive about 100 times amount administration greater than neurotrophin about 10.

99. according to the method for claim 97, wherein neurotrophin is selected from: NGF, BDNF, NT-3, NT-4 and NT-5.

100. according to the method for claim 97, wherein the p75 receptor-binding agents comprises neurotrophin binding motif or its peptide mimics.

101. according to the method for claim 97, wherein the p75 receptor-binding agents is cyclic peptide or the peptide mimics that comprises aminoacid sequence TDIKGKE (SEQ ID NO:42) in its ring.

102. according to the method for claim 97, wherein the p75 receptor-binding agents is N-Ac- CTDIKGKEC-NH ₂(SEQ ID NO:43).

103. method that promotes to suppress the CNS neure growth in the environment, this method comprises the p75 receptor-binding agents that gives the individual treatment effective dose, and wherein the p75 receptor-binding agents is not in conjunction with the neurotrophin by the Trk receptor of injured neurons expression in the inhibition environment.

104. according to the method for claim 103, the wherein p75 receptor-binding agents neurotrophin linked together administration different with other, wherein other different neurotrophin is in conjunction with the Trk receptor of being expressed by injured neurons in the inhibition environment.

105. according to the method for claim 104, wherein the p75 receptor-binding agents is NGF, and other different neurotrophin is BDNF.

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