CN101448513A - Methods and compositions related to improving properties of pharmacological agents targeting nervous system - Google Patents

Abstract

The present invention discloses compositions and methods related to improving pharmacological properties of bioactive compounds targeting nervous system.

Description

The method and composition relevant with the character of the pharmacological agents that improves targeting nervous system

CROSS-REFERENCE TO RELATED PATENT

The serial number that the application requires on January 5th, 2006 to submit to is that serial number that 60/757,047 U.S. Provisional Application and JIUYUE in 2006 were submitted on the 11st is the rights and interests of 60/844,024 U.S. Provisional Application.The serial number of submitting on January 5th, 2006 be serial number that 60/757,047 U.S. Provisional Application and JIUYUE in 2006 were submitted on the 11st be 60/844,024 U.S. Provisional Application full text all by reference mode include this paper in.

Background technology

Blood brain barrier (BBB) is separated mammiferous brain and body circulation, and plays crucial effects in the stable state of central nervous system (CNS).Although stride and constantly make progress aspect the transhipment of blood brain barrier understanding peptide, how they directly are delivered to effectively and remain a main difficult problem that exists in the process of exploitation as the neuropeptide of potential therapeutic agent among the CNS.

For example, epilepsy is a kind of neurological disorder of complexity.According to estimates, have 30% to suffer from intractable epilepsy among the epileptic.Although there have been various antiepileptics (AED), only there is a spot of AED in to the epilepsy of some type and the syndromic treatment of epilepsy, to obtain limited success.Therefore, still need to find and develop new convulsion medicine with improved effectiveness and safety.In addition, the discovery that changes about neurobiology before the epilepsy recently provides a chance of finding new epilepsy chemical compound, and this class Anti-epileptics can comprise neuropeptide and neurotrophic factor.

The neuropeptide and the receptor thereof that relate to epilepsy mechanism comprise galanin, neuropeptide tyrosine, somatostatin and opioid peptide.When in the above-mentioned neuropeptide some directly being delivered among the central nervous system (CNS), they have anti-convulsant activity, but its weak bioavailability and low metabolic stability have hindered the exploitation based on the antiepileptic of neuropeptide.On the other hand, Xian Jin peptide engineering has produced many successful cases with peptide analogues of improved stability or bioavailability.Yet, still be applied to having the neuropeptide of anti-convulsant activity without any a kind of feasible peptide engineering technology.What need in this area is the method and composition that is used to improve the permeability that passes blood brain barrier.

Summary of the invention

Concrete and the broadly described purpose of the present invention according to this paper, one aspect of the present invention relates to a kind of isolating polypeptide, described polypeptide comprises SEQ ID NO:3, have aminoacid sequence with the aminoacid sequence of SEQ ID NO:3, or have the aminoacid sequence of the metathetical SEQ ID of one or more conservative amino acid NO:3 at least about 90% homogeneity.

The invention also discloses a kind of isolating polypeptide, contain and be selected from SEQ ID NO:2,4-29,37-39,50,64,65,66,67,80,82 and 89 aminoacid sequence, with be selected from SEQ ID NO:2,4-29,37-39,50,64,65,66,67,80,82 and 89 aminoacid sequence has the aminoacid sequence at least about 90% homogeneity, perhaps has the metathetical SEQ of the being selected from ID of one or more conservative amino acid NO:2,4-29,37-39,50,64,65,65,66,67,80,82 and 89 aminoacid sequence.

The invention also discloses a kind of isolating polypeptide, contain the amino acid fragment that is selected from SEQ ID NO:31-36, have aminoacid sequence with the aminoacid sequence that is selected from SEQ ID NO:31-36, perhaps have the aminoacid sequence of the metathetical SEQ of the being selected from ID of one or more conservative amino acid NO:31-36 at least about 90% homogeneity.

The invention also discloses a kind of isolating polypeptide, comprise SEQ ID NO:40, have aminoacid sequence with the aminoacid sequence of SEQ ID NO:40, or have the aminoacid sequence of the metathetical SEQ ID of one or more conservative amino acid NO:40 at least about 90% homogeneity.

The invention also discloses a kind of isolating polypeptide, contain the amino acid fragment that is selected from SEQ ID NO:105,106,107,108,109-112 and 113-118, have aminoacid sequence with the aminoacid sequence that is selected from SEQ ID NO:105,106,107,108,109-112 and 113-118, perhaps have the aminoacid sequence of the metathetical SEQ of the being selected from ID of one or more conservative amino acid NO:105,106,107,108,109-112 and 113-118 at least about 90% homogeneity.

The invention also discloses a kind of isolating polypeptide, comprise the amino acid fragment that is selected from SEQ ID NO:58 and 135-141, have aminoacid sequence with the aminoacid sequence that is selected from SEQ ID NO:58 and 135-141, perhaps have the aminoacid sequence of metathetical SEQ IDNO:58 of one or more conservative amino acid and 135-141 at least about 90% homogeneity.

The invention also discloses a kind of compositions with enhanced blood-brain barrier permeability, wherein said compositions comprises a kind ofly compares the peptide with enhanced lipophilic characteristic and enhanced alkalescence with this peptide of not transformed.

The invention discloses a kind of method that increases the blood-brain barrier permeability of peptide, comprise comparing when making described peptide and not transformed to have enhanced lipophilic characteristic and alkalescence.

The invention also discloses a kind of method for the treatment of epilepsy, comprise the polypeptide disclosed herein of the experimenter's effective dose that needs treatment.The invention also discloses a kind of method for the treatment of epilepsy, comprise the compositions disclosed herein of the experimenter's effective dose that needs treatment.

The present invention has opened the method for a kind of treatment, prevention or alleviating pain or other neurological disorder, comprises the polypeptide disclosed herein of the experimenter's effective dose that needs treatment.

The invention also discloses a kind of method for the treatment of the experimenter of the compositions that need be able to pass blood brain barrier, comprising: differentiate the compositions that is used for the treatment of described experimenter; Modify described compositions by the lipotropy and the alkalescence that strengthen described compositions; And the experimenter who described modified compositions is needed treatment.

The invention also discloses a kind of method for the treatment of the experimenter of the compositions that need be able to pass blood brain barrier, comprising: differentiate the compositions that is used for the treatment of described experimenter; Modify described compositions by the lipotropy, glycosylation and the alkalescence that strengthen described compositions; And the experimenter who described modified compositions is needed treatment.

The invention also discloses a kind of method for the treatment of the experimenter of the compositions that need be able to pass blood brain barrier, comprising: differentiate the compositions that is used for the treatment of described experimenter; Modify described compositions by the lipotropy, glycosylation and the alkalescence that strengthen described compositions; Described modified compositions is inserted in the carrier; And the experimenter who described carrier is needed treatment.

The invention also discloses a kind of method for the treatment of the experimenter of the compositions that need be able to pass blood brain barrier, comprising: differentiate the compositions that is used for the treatment of described experimenter; Modify described compositions by the lipotropy and the alkalescence that strengthen described compositions; Described modified compositions is inserted in the carrier; And the experimenter who described carrier is needed treatment.

Description of drawings

Following accompanying drawing---is included into this description and constitutes its part---and shows several embodiments of the present invention, and is used from explanation principle of the present invention with description one.

Fig. 1 shows the effect of neuropeptide in the neurotransmission that is mediated by GABA and GLU in SSSE hippocampal neuron loop.Abbreviation: CA, pyramidal cell; DYN, dynorphin; GABA, γ-An Jidingsuan; GAL, galanin; GLU, glutamate, Glu; NE, norepinephrine; NPY, neuropeptide tyrosine; SOM, somatostatin; SubsP, P material (Wasterlain et al.2002).

Fig. 2 shows the persistent period that the galanin that was expelled in the district can shorten the rat epilepsy before or after stimulating.Last figure: penetrating fiber stimulation (PPS) injection in 30 minutes before galanin (50 and 500 picomole).Figure below: in 30 minutes whens injection after PPS, only need inject the persistent period (Mazarati et al.1998) that 500 picomole galanins just can effectively reduce epilepsy.

Fig. 3 shows the galanin receptors agonist of non-peptide and incubation period that galanin all can prolong mice and reduces the epilepsy scoring of the inductive epilepsy of PTZ.Illustration has been summarized maximum epilepsy scoring (black, contrast; Hollow, galnon; Lycoperdon polymorphum Vitt, galanin) (Saar et al.2002).

After Fig. 4 shows and inject somatostatin in giving self-sustaining attitude epilepsy model (self-sustaining statusepilepticus model) rat hippocampus, the anti-convulsant activity of somatostatin (Mazarati and Wasterlain, 2002).

After Fig. 5 shows and inject dynorphin and neuropeptide tyrosine in giving self-sustaining attitude epilepsy model rat hippocampus, their anti-convulsant activity.Control experiment please refer to Fig. 4 (Mazarti and Wasterlain, 2002).

Fig. 6 shows the key factor of the blood-brain barrier permeability that influences peptide.Alkalescence and lipotropy can improve the passive transport via the endocytosis of diffusion and absorption mediation, and glycosylation or carrier also can promote the active transport by blood brain barrier.

Fig. 7 shows the group method that is used to develop the neuropeptide analog with anti-convulsant activity.Select two kinds of model neuropeptides to assess the technology that is used to improve its blood-brain barrier permeability.

Fig. 8 shows a kind of structure organization of prototype of ideal neuropeptide medicine.The residue of described pharmacophore represented in asterisk.Metabolic stability, alkalescence and lipophilic main chain and the side chain of the described peptide moiety of gray frame representative can increasing are modified.Described BBB/PK-modified protein be one big, based on the structure of polymer, comprise lipophilic unit and cation unit and endogenous nutrition analogies.Described cation unit is by increasing the endocytosis that absorbs mediation with film generation electrostatic interaction.Described lipophilic unit can strengthen the passive transport by blood brain barrier.Active transport analogies structure (for example, hexose or phenylalanine) can be used as be positioned at blood brain barrier to the interactional substrate of nutrition transport protein.

Fig. 9 shows the method for the peptide analogues that is used to design penetrable BBB.Show the combination that two or more different chemicals that can improve described analog BBB permeability are modified.

Figure 10 shows and is used to transform neuropeptide to improve the system approach of its blood-brain barrier permeability.Key word: DAB, DAB; DAP, diaminopropionic acid; PEG, Polyethylene Glycol; Mmt, 4-methyl trityl.

Figure 11 shows the terminal cellular construction that extends of N-of somatostatin analogs.Described unit is link coupled in the solid-phase peptide building-up process.Unitary quantity and order are arbitrarily, and can optimize BBB/PK and regulate proteic structure composition.

Figure 12 shows Gal BBB-2 demonstrates time (illustration) and the dependent anti-AGS of dosage in Frings mice body protective effect.During this period, use the Gal BBB-2 processing mice and the different time points after administration of 4mg/kg peritoneal injection that it is tested.Use the Gal BBB-2 of cumulative dosage to handle to be used for the Frings mice of dose response study group and after the peritoneal injection administration, it was tested in 1 hour.

Figure 13 shows the overall experimental technique of optimizing GAL-BBB2.First three block diagram has been summed up the activity of definition among the embodiment 1.Synthesized near 40 kinds of analog and with competitive binding assay and screened.Before carrying out more detailed pharmacology's sign, only the most effective galanin part is screened with regard to effectively reaching long lasting anti-convulsant activity.

GAL-BBB2 structure organization that Figure 14 shows and the SAR that is proposed research.Black surround shows crucial pharmacophore residue.

Figure 15 shows " main chain prosthese "-with non-peptide sept (5-aminovaleric acid) displacement non-pharmacophore residue (R2 and R3).Other main chain septs comprise aminocaproic acid or amino-3,6-two oxa-s sad (PEG-sept).

Figure 16 show GAL-BBB2 (being labeled as NAX-5055 among the figure) during the inflammation phase of initial acute stage and prolongation but (0.52-5mg/kg) dose dependent reduce and lick foot.On the contrary, find that the natural fragment Gal-16 of unmodified after the peritoneal injection administration is a non-activity, its dosage is 4 times (being 20mg/kg) of the GAL-BBB2 maximum dose level tested.

The GAL-BBB2 that Figure 17 shows 5mg/kg is equivalent to the gabapentin of 10mg/kg dosage.

Figure 18 shows in the sciatic nerve ligation model of chronic pain, and GAL-BBB2 (being labeled as NAX-5055 among the figure) makes the super quick threshold value of the mechanicalness pain sensation demonstrate the increase of time dependence.In addition, GAL-BBB2 is equivalent to morphine and than the high several times of effectiveness of gabapentin.

Figure 19 shows the structure of GAL-BBB2 (NAX 5055 is otherwise known as).

Figure 20 shows NAX 5055 (GAL-BBB2) in Frings mice body but not the native peptides fragment is activated.In dose response study, the quantitative convulsion usefulness of (1 hour) during peak action.The result of this research shows that GAL-BBB2 demonstrates the effect of the anti-AGS of dose dependent.Median effective dose that is calculated (being ED50) and 95% confidence interval obtain by the dose response data being carried out probit analysis, and described dosage is 3.2 (2.3-6.1) mg/kg.Described native peptides fragment GAL (1-16) is non-activity (GAL-BBB2 ED50 6 times) when the dosage with 20mg/kg carries out peritoneal injection.

Figure 21 shows chemical constitution and the sketch map of transforming octreotide.

Figure 22 shows tiring of in 6Hz (32mA) test NAX 5055 (GAL-BBB2) and usefulness all is higher than native peptides.

Figure 23 shows NAX 5055 (GAL-BBB2), and (4mg/kg i.p.) demonstrates the anti-convulsant activity of time dependence in Frings mice body.

Figure 24 shows NAX 5055 (GAL-BBB2) demonstrates the anti-AGS of dose dependent in Fr ings mice body protective effect.

It is activated in drug resistant epilepsy outbreak model (4mg/kg) that Figure 25 shows NAX 5055 (GAL-BBB2).

Figure 26 shows after i.p. and s.c. administration, and NAX 5055 (GAL-BBB2) demonstrates good bioavailability in the test of 6Hz epilepsy.With NAX 5055 intraperitoneal or be subcutaneously injected into each the group (n=6-8) male CF-1 mice body in.After 60 minutes, each mice in every group all stimulates (32mA, 6Hz last 3 seconds) with Corneal electrode.Think that the mice that does not show the limbic lobe epilepsy all is to be subjected to protection.The result shows that the anti-convulsant activity of NAX 5055 has obtained keeping after subcutaneous administration.These discoveries show can use the storage dosage form that is suitable for subcutaneous delivery NAX 5055 and/or other neuroactive peptides.

Figure 27 shows in 6Hz (44mA) test, and NAX 5055 (GAL-BBB2) can increase the usefulness of CMPDX and tire.When giving separately, the CMPD A (levetiracetam) of higher dosage suppresses 6Hz limbic lobe epilepsy when 1000mg/kg (that is, protective effect the highest by 50%) with minimum usefulness.On the contrary, when with the NAX5055 (1.5mg/kg) of minimum effective dose and CMPDA A (levetiracetam) when giving jointly, usefulness and tiring is significantly increased.The above results shows that the modification of 5055 pairs of galanin receptors of NAX can cause the convulsion usefulness concertedness of levetiracetam to strengthen.When giving jointly, above-mentioned discovery shows with the levetiracetam of independent higher dosage to be compared, and what combine NAX 5055 and levetiracetam has more the treatment advantage in conjunction with product.

Figure 28 shows the protective effect that NAX 5055 (GAL-BBB2) (hippocampalkindled rat) in Hippocampus kindled rats body demonstrates moderate.In the Hippocampus kindled rats model of partial epilepsy, NAX 5055 is reduced to 3 with the epilepsy scoring from 5.These results show that the adjusting of 5055 pairs of galanin receptors of NAX can be used for preventing and treating secondary general part epilepsy, and the Intraventricular result of study formerly that is injected directly in the kindled rats brain with galanin wherein is consistent.The NAX5055 of intraperitoneal administration is that activated this discovery has proved following conclusion, and promptly NAX 5055 has entered brain after the whole body administration.

Figure 29 shows the inductive hyperalgesic effect of NAX 5055 (GAL-BBB2) PARA FORMALDEHYDE PRILLS(91,95).Before vola injection formaldehyde, give NAX 5,055 60 minutes the time.The convulsion time when time is based on peak action.

Figure 30 shows somatostatin and δ-sleep inducing peptide (DSIP) at 22mA, all can be used as anticonvulsant in the test of 6Hz epilepsy.Result shown in this figure proof is when in the ventricles of the brain space that somatostatin and δ-sleep inducing peptide (DSIP) directly is administered to the CF-1 mice, and they can effectively suppress 6Hz (22mA) limbic lobe epilepsy.The above results provides a kind of like this " Proof of Concept ", and promptly the adjusting to somatostatin in the brain and DSIP binding site is a kind of practicable approach.They also help to use our patented technology exploitation to have systemically active somatostatin and the DSIP neuroactive peptide that passes blood brain barrier.

Figure 31 shows the structure of GAL (1-16) analog.The part of labelling is the critical information fragment that can be used for designing the analog with enhanced BBB permeability.

Figure 32 shows the NAX-5055 that injects twice every day, and (4mg/kg i.p.) lights the influence of acquisition to the mice cornea.The CF#1 mice is accepted carrier (0.9% saline) or NAX-5055 at random.Mice in the NAX-5055 group is at its NAX-5055 that is subjected to lighting 12 hours (12h) before stimulating first and respectively accepted dose in 1 hour.Carry out 1 hour (1h) before the subsequent stimuli at every turn, the mice of NAX-5055 processed group accept another time dosage NAX-5055 (4mg/kg, i.p.).Stimulate mice twice every day, continue 16 days.The result is expressed as the average epilepsy scoring of each stimulation.As mentioned above, the result of NAX-5055 processing mice is divided into two groups: promptly responsive organize (green line) and insensitive group (blue line).There is significant difference in p＜0.0002 o'clock between the responsive group of saline group and NAX-5055; There is significant difference in p＜0.0001 o'clock between insensitive group of responsive group of NAX-5055 and the NAX-5055.The result that this research obtains has supported following conclusion, promptly has the ability that obtains to light of preventing based on the peptide (for example NAX-5055) of galanin, and can improve the state of an illness in patient's body of easily suffering from epilepsy and other neurological disorderes.

Figure 33 show use every day NAX-5055 to handle twice (mg/kg, i.p.) corneal is lighted the influence of speed.The mice (experimental detail is referring to the caption of figure X) of handling with NAX-5055 is divided into two processed group: promptly responsive group of NAX-5055 and NAX-5055 are insensitive group.The result be expressed as need reach the scoring of specific epilepsy+/-the required stimulation quantity of S.E.M (being 1-5).The mice of the responsive group of NAX-5055 needs the stimulation more than 2 times to reach the 1st grade of epilepsy, and needs the above stimulation of 35-40% to reach the 2nd grade and 3rd level epilepsy respectively.In addition, the mice in the responsive group of NAX-5055 does not all reach the 5th grade of epilepsy.One factor analysis of variance, p＜0.0209; Factor analysis: saline group and NAX-5055 compare p for insensitive group〉0.05; The responsive group of NAX-5055 compares p＜0.05 for insensitive group with NAX-5055.The above results is supported such conclusion, be that the modified neuropeptide based on galanin has the ability of lighting the development of obtaining (kindling acquisition) that changes, and they can be used to prevent the network hyperexcitability in the patient group who easily suffers from epilepsy and other neurological disorderes.

Figure 34 shows the structure (Elmagbari, Egleton et al.2004) that is incorporated into the glycosyl group in the ICI154129 N,N-bisallyl-Tyr-Gly-Gly-.

The combination that Figure 35 shows two kinds of different chemicals modifications is better than independent modification.Independent cationization or fatization can not improve the permeability of described 5055 analog as both combinations.

Figure 36 shows the example of the lipoamino acid of the blood-brain barrier permeability that is used to improve peptide.This class lipoamino acid can be united with the chemical modification of the alkalescence that strengthens described target peptide.

Figure 37 shows the metabolic stability that chemical modification has improved described neuropeptide analog.The analog Gal (1-16) of analog 5055 (SEQ ID NO:3) or 1205-2 (SEQ ID NO:50) or unmodified is hatched in the rat blood serum that is diluting under 37 ℃.Utilize HPLC to measure the surplus of described peptide.

Figure 38 shows dissimilar neuropathic pains and can utilize the neuropeptide analog that can stride across BBB to treat, prevent or reverse.

The specific embodiment

With reference to following to the detailed description of the preferred embodiments of the invention and wherein included embodiment and with reference to the accompanying drawings and front and back literary composition description can understand the present invention more easily.

Before disclosure and description chemical compound of the present invention, compositions, goods, equipment and/or method, it should be understood that except as otherwise noted, the invention is not restricted to specific synthetic method, specific reorganization biological technique method; Perhaps except as otherwise noted, the invention is not restricted to specific reagent, yes in aspect hereinafter described itself can change.It should also be understood that term used herein only is is not to be intended to limit in order to describe specific embodiment.

A. definition

Unless spell out in addition in the context, the singulative that uses in this description and the claims " ", " one " and " being somebody's turn to do " comprise that plural number refers to object.Comprise mixture of two or more this carriers or the like when therefore, for example, relating to " pharmaceutical carrier ".

Scope can be expressed as from " approximately " concrete numerical value herein, and/or to " approximately " another concrete numerical value.When being expressed as this scope, another embodiment comprises from a described concrete numerical value and/or to described another concrete numerical value.Similarly, when " approximately " by using the front numeric representation during as approximation, be should be understood that this concrete numerical value constitutes another embodiment.Should be understood that further that the end points of each scope is relevant with another end points and all be significant when being independent of another end points.It is to be further understood that to herein disclosed is many numerical value, and each numerical value is also open with " approximately " this concrete numerical value form at this except that described numerical value itself.For example, if disclose numerical value " 10 ", " about 10 " are disclosed so also.It is to be further understood that when disclosing a numerical value, also disclose the possible range between " being less than or equal to " this numerical value, " more than or equal to this numerical value " and numerical value, this as those skilled in the art appropriate understand.For example, if disclose numerical value " 10 ", " being less than or equal to 10 " and " more than or equal to 10 " are disclosed so also.

In this description and claims, will be referred to many terms that should be defined as having following implication:

" optional " or " randomly " means incident or the situation described subsequently and may maybe can not exist, and such description comprises situation and described incident or the non-existent situation of situation that described incident or situation exist.

B. compositions and method

1. blood brain barrier

Blood brain barrier (BBB) is the specialization system of capillary endothelial cell, and the harmful substance that described system can protect brain to exempt from the blood flow is attacked, and exercises the required nutrition of normal function for brain provides simultaneously.Relatively freely the periphery blood capillary of exchange is different with allowing material to stride cell/between cell, the strict restricted passage physics of BBB (closely connecting) barrier and metabolism (enzyme) barrier go into vertigo fortune.Therefore, BBB normally determines medicine to go into the rate-limiting factor of brain infiltration.

Present many stops limit chemical compound lot be used as central nervous system (CNS) therapeutic agent.At first, brain possesses barrier system.Brain barrier system has two primary clusterings: choroid plexus and blood brain barrier (BBB).Choroid plexus is separated cerebrospinal fluid (CSF) with blood, BBB separates brain ISF with blood.

In addition, much about 1000 times of the surface area ratio choroid plexus of BBB, and be the major obstacle that the treatment chemical compound is delivered to CNS.BBB plays the effect of selecting to distribute, regulate material (comprising peptide) exchange between CNS and peripheral circulation.The primary structure of BBB is the brain capillary endothelial cell wall.The tight connection of brain brain capillary endothelial cell can prevent that the circulation chemical compound from arriving brain ISF by the cell bypass approach.In addition, nearest studies show that the barrier that provides except that tight connection, also has physiologic barrier (Kroll et al., Neurosurgery, Vol.42, No.5, p.1083 (May1998)) in the basal layer level.Other specific characteristics of BBB comprise on the surface, chamber of endothelium and lack intracellular membrane hole, pinocytotic vesicle and net negative charge.

Material can be divided into active transport mechanism and passive transport mechanism usually so as to the mechanism of passing BBB.The easy utilization of lipophilic molecule can be passed BBB by the passive transport or the diffusion of endothelium plasma membrane.Hydrophilic molecules (for example peptide) needs the active transport system to make them can pass BBB usually.Some bigger peptide (for example insulin) has the lip-deep receptor in the chamber that is positioned at the brain blood capillary, and described brain blood capillary plays the initiatively effect of dysuria with lower abdominal colic system.

Have two kinds of main mechanism of peptide being transported blood brain barrier: (1) simply strides the film diffusion, mainly determined by molecular size and lipotropy, (2) active absorption, mediated by the specific receptor and the carrier that are arranged on the blood brain barrier endothelial cell surface, perhaps mediated (Tamai and Tsuji, 2000 by non-specific absorbability dysuria with lower abdominal colic; Pan and Kastin, 2004a; Smith et al., 2004).

2. raising permeability

After tested many methods improve peptide and proteic blood-brain barrier permeability.These methods can be divided into following a few class: (1) with can strengthen CNS to the carrier of the picked-up of this peptide or puting together of nutriment active transport body (nutrient-active transport); (2) can produce enhanced lipotropy to strengthen the fatization/halogenation of simple diffusion; (3) can strengthen the cationization (alkalescence of increase) of the transhipment of carrying out via the absorbability dysuria with lower abdominal colic; (4) can improve the active transhipment of peptide and glycosylation or the prodrug ((Witt et al., 2001) and (Pan and Kastin, 2004a)) that drug metabolism distributes.This class research and the main example of finding are shown in the table 1.Every piece of list of references is included this description in about the whole modes by reference of the instruction that penetrates blood brain barrier.

Table 1. improves the example of the blood-brain barrier permeability of peptide

Method	Peptide	The main discovery	List of references
				The halogenation of aromatic moieties	Enkephalin	The peptide that contains chloro-Phe behind the intravenously administrable can produce stronger analgesic activity	(Weber et al.， 1991；Abbruscato et al.，1996)
Put together with lipoamino acid	Alpha-conotoxin MII	Blood-brain barrier permeability increases by 4 times	(Blanchfield etal.，2003)
				Put together with polyamine derivative	The amyloid beta derived peptide	Blood-brain barrier permeability increases by 7 times	(Poduslo et al.，1999)
Put together with the DOPA/DTPA derivant	Octreotide/somatostatin epidermal growth factor	The brain capture of radiopharmaceuticals that is used for the MRI of the cerebral tumor and treatment is more effective	(Luyken et al.， 1994；Kurihara and Pardridge，1999)
				Glycosylation	Enkephalin	Add that glycosyl part can cause systemic bioavailability to increase by 20 times	(Elmagbari et al.，2004)
The displacement of selectivity amino acid side chain	Neurotensin	Trp is replaced by Neo-Trp and Ile is put by Tert-Leu	(Hertel et al.，2001)

		Change and to produce the analog that can pass blood brain barrier
				The displacement of selectivity amino acid side chain	Throtropin releasing hormone (TRH)	His can be made the active increase of TRH maincenter 2-3 doubly by the pyridine moiety displacement	(Prokai et al.，2004)
The prodrug method	TRH sample peptide	The esterification meeting of Glu residue strengthens the effect of reviving	(Prokai-Tatrai etal.，2003)
				The prodrug method	Enkephalin	The C-terminal extension that has Phe can increase half-life and permeability	(Greene et al.，1996)
The prodrug method	Enkephalin	Put together with diamantane (obsolete) part (adamantine moiety) and can make active rising the when the subcutaneous administration	(Kitagawa et al.，1997)
				Carrier mediated sends	The enkephalin Dalargin	Carrier based on the SynB3 peptide can strengthen brain capture and sedation	(Rousselle et al.，2003)

About first, put together, as if the increase of puting together the peptide molecule size do not hinder permeability.Go up addition peptidyl carrier S ynB1 (MW=2,099) at opioid peptide Dalargin (MW=726) and can make size increase almost 4 times, but also make brain capture increase by 18 times (Rousselle et al., 2003).Similarly, can behind intravenously administrable, increase its anti-injury activity at two sugar moieties of addition on the ICI154129 N,N-bisallyl-Tyr-Gly-Gly-and be up to 21 times (Elmagbari et al., 2004).

---to be lipotropy and alkalescence---help to increase the blood-brain barrier permeability of peptide, and do not need unitransport albumen or carrier two important factors.The lipophilic characteristic of peptide can be by puting together with hydrophobic part (for example, poly-aliphatic chain) or aromatic moieties being carried out halogenation (for example, comparing with Phe, is exactly chloro-Phe) transform.Verifiedly compare with peptide with the albumen of unmodified, albumen and peptide that polyamines is modified can more effectively pass blood brain barrier (Poduslo and Curran, 1996a; B; Poduslo et al., 1999).The alkalescence of the increase of also verified (Tamai et al., 1997) little peptide is an important determiner of striding the blood brain barrier transhipment via the endocytosis (AME) that absorbs mediation.

Except that direct modified peptides, also have some can strengthen the delivery method of CNS to the picked-up of medicine.These methods comprise, and the blood brain barrier of striding liposome-mediated, the micelle mediation or the peptide that nano-particle mediates is sent (Kreuter et al., 2003; Pan and Kastin, 2004b).These new medicine delivery techniques can also be used for the compositions based on neuropeptide disclosed herein and known in the art.

The peptide engineering method that has several blood-brain barrier permeabilities that can improve neuropeptide.Among Fig. 6 these methods are summarized.Yet research mentioned above does not provide in conjunction with the system approach of said method with the blood-brain barrier permeability of the described peptide of further enhancing.Disclosed herein is that above-mentioned peptide engineering method and known convulsion neuropeptide (for example somatostatin or galanin) are used in combination, thereby makes above-mentioned peptide bring into play anti-convulsant activity via intravenous (i.v.) or subcutaneous (s.c.) route of administration.

Disclosed herein is the method and composition that is used to increase blood-brain barrier permeability." increase " is meant that the described compositions of higher proportion can be passed blood brain barrier with wild type, that do not transform or native peptides is compared or compare with reference composition.For example, with contrast, peptide natural or wild type or compositions compare, the ratio of increase can be percent 1,2,3,4,5,

6,7,8,9,10,11,12,13,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,125,150,175,200,225,250,275,300,325,350,375,400,425,450,475,500,550,600,650,700,750,800,850,900,950,1000,1250,1500,1750,2000,2250,2500,2750,3000,3500, or 4000.

Particularly, the invention discloses a kind of compositions with enhanced blood-brain barrier permeability, wherein said compositions comprises a kind of peptide (Fig. 7) with enhanced lipophilic characteristic and enhanced alkalescence of comparing with this peptide of not transformed.The invention also discloses compositions with enhanced blood-brain barrier permeability, wherein said compositions comprise a kind of compare with this peptide of not transformed have enhanced lipophilic characteristic, enhanced alkalescence and enhanced glycosylated peptide.

The invention also discloses the method for the blood-brain barrier permeability that strengthens peptide, comprise making described peptide have enhanced lipophilic characteristic and enhanced alkalescence with not compared by the peptide of forms of modification.The another kind of method that increases the blood brain barrier penetration power of peptide comprises making described peptide have enhanced lipophilic characteristic, enhanced alkalescence and enhanced glycosylation with not compared by the peptide of forms of modification.

A) lipophilic characteristic

For example, the lipophilic characteristic of described compositions can be strengthened by described peptide and a kind of hydrophobic part are puted together.The example of hydrophobic part includes but not limited to poly-aliphatic chain or aromatic moieties.Described lipophilic characteristic can also be strengthened by the halogenation that increases aromatic moieties or poly-aliphatic chain reagent (for example perfluor caproic acid).

B) alkalescence

The alkalescence of described compositions can be strengthened by same oligomer and the different oligomer of introducing the positive electricity amino acid residue, and described positive electricity amino acid residue includes but not limited to the ornithine of lysine, arginine, high-lysine (homo-lysine), homoarginine, L-or D-isomer configuration; 2, the 3-diaminopropionic acid; 2,4-diamino-butanoic.

Alkalescence also can be by being strengthened with puting together based on the part (polyamine-based moiety) of polyamines, and described part based on polyamines is spermine, spermidine, polyamide-amide type dendrimer or polyamines toxin for example, and their derivant.

C) glycosylation

Glycosylation can be introduced by puting together with xylose, glucose, galactose, maltose, maltotriose, mannose, lactose, 6-(.alpha.-D-galactosido)-D-glucose. or similar saccharide.

D) carrier

The invention also discloses the carrier that contains compositions disclosed herein.At Toyobuku et al. (J Pharmacol Exp Ther.2003 Apr; 305 (1): the example that can find the carrier that can pass BBB 40-7.).

C. Therapeutic Method

The invention discloses the treatment specified disease relevant and the method for obstacle, perhaps anyly relate to the application of compound that needs can pass blood brain barrier with the central nervous system.Use method and composition disclosed herein can treat various diseases and obstacle, comprise apoplexy and relevant ischemic diseases, chronic back pain, spinal cord injury, peripheral nerve injury, traumatic brain injury, retinal degeneration, nerve retrograde affection, cataract, antibiotic-associated ototoxicity, Alzheimer, amyotrophic lateral sclerosis (ALS, Lou Gehrig disease), epilepsy (generalized epilepsy for example, partial epilepsy or intractable epilepsy), the Heng Yandunshi chorea, parkinson, multiple sclerosis, chronic back pain, fibromyalgia, postherpetic neuralgia, diabetic neuropathy, traumatic mononeuropathy, the complex region pain syndrome, auxiliary analgesic (adjuvant analgesic), spinal nerve root cut-out/neural cutting, preemptive analgesia/amputation, epilepsy forms (epileptogenesis)/wound, Chemical exposure, status epilepticus, the neuropathy of phase chemotherapy induced, cancer, opioid withdrawal and chronic neuropathic pain.Medication and approach, dosage and pharmaceutical composition have more detailed discussion hereinafter.

About using combination treatment spinal cord injury disclosed herein and multiple sclerosis, following list of references is included this description in about the whole modes by reference of the instruction for the treatment of above-mentioned disease: Hawes JJ, and Narasimhaiah R,

Picciotto MR Galanin and galanin-like peptide modulate neurite outgrowth via proteinkinase C-mediated activation of extracellular signal-related kinase.Eur J Neurosci.2006Jun；23(11)：2937-46.Suarez V，et al，The axotomy-induced neuropeptides galanin andpituitary adenylate cyclase-activating peptide promote axonal sprouting of primary afferentand cranial motor neurons Eur J Neurosci.2006 Sep；24(6)：1555-64。

The prolactin antagonist that compositions disclosed herein and method can also be used for the infringement of pre-oxygen deficit proof, increase the intravital growth hormone secretion of people, the regulation and control pituitary adenoma discharges, prolong the analgesic activity of morphine, as antidepressants, and be used for for example eating disorders.

The invention also discloses the method for treatment pain and other neurological disorderes, comprise the polypeptide disclosed herein of the experimenter's effective dose that needs treatment.

Method and composition disclosed herein can also be used for prevention, alleviate or the treatment neurological disorder those neurological disorderes for example disclosed herein and well known by persons skilled in the art.

Method and composition disclosed herein can be used in combination with other compositionss or Therapeutic Method.For example, following medicine and medicine class can be united with compositions disclosed herein and be used for pain, epilepsy, neuroprotective, depression, biphasic or bipolar type, other mental disorders, and the disease or the obstacle that are used for any other available combination treatment disclosed herein: opioid and opioid peptide, morphine, hydroxyl morphine (hydroxymorphine), fentanyl, oxycodone, codeine; Capsaicin; And antiepileptic class, include but not limited to carbamazepine, primidone, gabapentin, pregabalin, diazepam, felbamate, fluoro felbamate (fluorofelbamate), lamotrigine, lacosamide, levetiracetam, phenobarbital, phenytoin, fosphenytoin, topiramate, valproate, vigabatrin, zonisamide, oxcarbazepine, non-steroidal antiinflammatory drugs (NSAID), local anesthetic (for example lignocaine), glutamate receptor antagonist, nmda antagonist, alpha-2-adrenoceptor agonist and antagonist, adenosine, Cannabinoids, NK-1 antagonist (CI-1021), antidepressants (amitriptyline for example, desipramine, imipramine), the analog of galanin and derivant, somatostatin, δ-sleep inducing peptide, enkephalin, oxytocin, Pancreozymin, calcitonin, hydrocortisone hormone inhibin (cortistatin), nociceptin and other therapeutic agent, and pluronic P85 block copolymer based on neuropeptide.

Following medicine and medicine class can be united with compositions disclosed herein and be used for Alzheimer: amyloid depressant, for example Flurizan; Galantamine (Razadyne); Profit is cut down this bright (Exelon); Donepezil (aricept); Tacrine (romotal); Memantine (Namenda) and be used for the vaccine of Alzheimer.The invention also discloses and be used for the treatment of experimenter's the method that needs can pass the compositions of blood brain barrier, comprising: differentiate the compositions that is used for the treatment of described experimenter; Modify described compositions by the lipotropy and the alkalescence that strengthen described compositions; And with described modified compositions need the treatment the experimenter.

" combination " is meant except that compositions disclosed herein, can also the compositions that one or more are extra gives described experimenter.Above-mentioned composition can

The invention also discloses treatment needs to pass experimenter's the method for the compositions of blood brain barrier, comprising: differentiate the compositions that is used for the treatment of described experimenter; Modify described compositions by the lipotropy, glycosylation and the alkalescence that strengthen described compositions; And the experimenter who described modified compositions is needed treatment.

The invention also discloses and be used for the treatment of experimenter's the method that needs can pass the compositions of blood brain barrier, comprising: differentiate the compositions that is used for the treatment of described experimenter; Modify described compositions by the lipotropy, glycosylation and the alkalescence that strengthen described compositions; Described modified compositions is inserted in the carrier; The experimenter who described carrier is needed treatment.

The invention also discloses a kind of method for the treatment of the experimenter of the compositions that need be able to pass blood brain barrier, comprising: differentiate the compositions that is used for the treatment of described experimenter; Modify described compositions by the lipotropy and the alkalescence that strengthen described compositions; Described modified compositions is inserted in the carrier; And the experimenter who described carrier is needed treatment.

1. use the method for described compositions as research tool

Disclosed compositions can be used as research tool in every way.For example, disclosed compositions (as SEQ ID NO:1-55) can be as the reagent of for example studying epilepsy.

For example, described compositions can be used as the molecule that target has required function character with separation in combinatorial chemistry scheme or other screening schemes, for example galanin agonist or antagonist or partial agonist.

Described compositions can be used for finding that independent interaction and the network type between different neuropeptides in the nervous system, other neurotransmitteies, receptor and ion are usually interacts.For example, disclosed compositions can be used for the galanin receptors antagonist and can act on synergism between the medicine of the molecular target of expressing on the same neuron.The positive drug-drug interactions of this class is useful, because they can improve usefulness or/and the safety of treatment when two kinds of medication combined application.

As described herein, disclosed compositions can be as the reagent in the microarray or as the reagent of surveying or analyze existing microarray.Disclosed compositions can be used in any known method that is used for separating or differentiating single nucleotide polymorphism.Described compositions can also be used in any known screening experiment (relating to chip/microarray) method.The machine readable example that described compositions can also be used in the disclosed compositions of any known use is for example studied in the method for molecule modeling analysis that dependency or execution relate to disclosed compositions.

2. genetic modification method and gene interrupt method

Disclosed compositions and method can be used for carrying out the target gene interruption and modify in any animal body, described animal can be stood above-mentioned incident.For example, can change the gene that produces galanin has enhanced blood-brain barrier permeability with expression galanin analog.Genetic modification and gene interrupt being meant in animal body that (for example mammal) is method, technology and the compositions at center with selective removal or modifying gene or chromosome segment, and mode is to propagate described modification for the kind by described animal.Usually, for example described herein, use the carrier that is designed for specific chromosomal region homologous recombination contained in the cell to come the described cell of transfection.This homologous recombination incident can produce the have introducing chromosome of DNA of (for example in frame), and described DNA is an external source for DNA on every side.This class scheme can be incorporated into very specific sudden change (for example point mutation) in the contained genome of this cell.Herein disclosed is the method for carrying out this class homologous recombination.

One of preferred feature of carrying out homologous recombination in mammalian cell is that described cell should be able to be cultivated, because the occurrence frequency of required recombination event is lower.

In case described cell is produced by method as herein described, then can produce animal by stem cells technology or this cell of clone technology cause.For example, if transfection the cell of described nucleic acid be the stem cell of organism, then this cell can be used to produce the organism that its germ line cell contains described genetic modification or interruption in transfection with after cultivating, described then organism can be used to produce another animal that has described genetic modification or interruption in its whole cells again.Be used for producing the method that has the animal of described genetic modification or interruption in its whole cells at other, can use clone technology.These technology are extracted described nucleus through transfectional cell usually, and merge through cells transfected nuclear and oocyte described by merging or replacing, and can operate to produce an animal described oocyte then.Use clone technology replace the advantage of the method for ES technology be can the non-ES cell of transfection cell.For example, the fibroblast that is very easy to cultivate can be used as transfected cell, and genetic modification or gene interrupt event have taken place in the described cell, and the cell that is derived from this cell then can be used to clone whole animal.

D. compositions

Except compositions itself, the invention discloses and can be used for preparing the component of compositions disclosed herein and can be used in component in the method disclosed herein.Herein disclosed is above-mentioned substance and other materials, and it should be understood that when the combination that discloses above-mentioned material, subclass, interaction, group etc., and may openly clearly specifically be meant simultaneously above-claimed cpd various independent and common combination and permutation each the time, the present invention specifically considers and has described each situation.For example, if disclose and a kind of concrete galanin analog be discussed and many modifications that many molecules (comprising variant) are carried out have been discussed, unless then clearly state on the contrary, the present invention has considered each and every kind of combination and permutation and possible modification of described galanin analog clearly.Therefore, if disclose molecule A, B and C and molecule D, E and F, and the example A-D of a combination molecule is disclosed, although then do not enumerate every kind of situation individually, still considered every kind of situation individually and jointly, promptly considered to disclose combination: A-E, A-F, B-D, B-E, B-F, C-D, C-E and C-F.The arbitrary subclass or the combination of above-mentioned substance are also disclosed similarly.Therefore, for example, consider to disclose subgroup A-E, B-F and C-E.This notion is applicable to all aspects of the application, includes but not limited to prepare and use the step in the disclosed method for compositions.Therefore, can be the additional step of implementing if exist many, it should be understood that each step of above-mentioned additional step goes for any specific embodiment of disclosed method or the combination of embodiment.

1. the compositions relevant with blood-brain barrier permeability

The automatic epilepsy outbreak is what to be caused by the overwrought neuron over-discharge that mainly is arranged in Hippocampus.The nicergoline overbalance is used the inhibition mechanism of γ-An Jidingsuan (GABA) and is regulated and control epilepsy (Wasterlain et al., 2002) by the excitatory mechanism of glutamic acid mediation.Neurotransmission is adjusted by many endogenous neuropeptides, comprises neuropeptide tyrosine, galanin, nociceptin/orphanin and interior morphine peptide-1 (endomorphin-1).Fig. 1 shows possible association between glutamic acid, GABA and the neuropeptide in keeping the hippocampal neuron loop of epilepsy state certainly.

The effect of above-mentioned neuropeptide has used pharmacology and heredity (knock out or cross and express) method to be elaborated.For example, Oberto and colleague thereof (Oberto et al., 2001) use transgenic mice to characterize the interaction (also this being summarized in (Eva et al., 2004)) between GABA energy system, neuropeptide tyrosine and neuropeptide tyrosine (1) receptor in the corpus amygdaloideum.Similarly, Leresche and colleague thereof (Leresche et al., 2000) prove that somatostatin suppresses the neurotransmission of GABA mediation via presynaptic mechanism.The glutamic acid of having studied the galanin adjustment that is subjected to Hippocampus in two kinds of transgene mouse models (galanin knocks out (GalKO) and crosses the mice of expressing (GalOE)) discharges (Mazarati et al., 2000).In GalKO and GalOE mice, utilize the galanin that gives at maincenter to increase and reduce the inductive glutamic acid of depolarization respectively and discharge, show that the galanin of Hippocampus plays the effect (Mazarati, 2004) of anticonvulsant by glutamatergic system.

Proved and had at least three kinds of neuropeptides and receptor thereof in epilepsy invasion, to work: galanin, somatostatin and neuropeptide tyrosine.After edge system presented status epilepticus, the immune response activity of the galanin in the Hippocampus descended.The injection galanin can prevent that the edge from being the morbidity of status epilepticus and stopping status epilepticus in hippocampal dentate door district.Therefore, as if galanin can be used as the endogenous anticonvulsant (Mazarati et al., 1998) that suppresses status epilepticus.Cross the phenotype of the transgenic mice of expression and confirmed this point (Kokaia et al., 2001) by checking galanin.In this research, galanin has suppressed kindling (kindling) epilepsy invasion.

Have realized that neuropeptide role in adjusting neurotransmitter release and epilepsy regulation and control can be used for developing new Therapeutic Method.As mentioned below, many disclosed neuropeptides that studies show that have effective anti-convulsant activity in animal model.

A) neuropeptide tyrosine and dynorphin

Neuropeptide tyrosine can suppress epileptic activity (Klapstein andColmers, 1997) in the external rat hippocampus section.In another research (Baraban et al., 1997), the mice of shortage neuropeptide tyrosine is uncontrollable epilepsy because of kainic acid produces.In addition, 93% knock-out mice can be dead gradually, and death condition is rarely found in the wild type animal.The Intraventricular neuropeptide tyrosine can suppress the death brought out owing to the kainic acid administration.Finally, the anticonvulsant action of confirmation neuropeptide tyrosine can mediate (Sperk and Herzog, 1997) via the Y5 receptor.

Confirmed to relate in epilepsy invasion and the epilepsy Hippocampus opioid peptide (comprising dynorphin) (in (Hong, 1992) and (Solbrigand Koob, 2004) this being summarized).Light the early stage release that the epilepsy that brings out can cause enkephalin and dynorphin by ECS or corpus amygdaloideum, but also can cause the long-term reduction (Gall, 1988) of dynorphin.As shown in Figure 5, dynorphin demonstrates anti-convulsant activity (Mazarati and Wasterlain, 2002) in the rat model of keeping the epilepsy state certainly.

The same with dynorphin, the neuropeptide tyrosine of (i.h.) injection also can reduce from the epilepsy distribution of keeping in the epilepsy state model (Mazarati and Wasterlain, 2002) in the Hippocampus.Be administered to the interior NPY of tricorn a kind of effective inhibitor of the inductive epilepsy of kainate (Woldbye etal., 1997) seemingly.The someone thinks antiepileptic action by neuropeptide Y 5 receptor mediation, and that uses that the Y5-deficient mice carries out studies confirm that this discovery (Marsh et al., 1999).

B) galanin and analog thereof

Because the work of Mazarati and coworkers has realized that galanin is a kind of effective anticonvulsant (Mazarati et al., 1992).When being injected directly into galanin in the tricorn or in the Hippocampus time, galanin can reduce the order of severity that Picrotoxin brings out in the rat body kindling is fainted from fear.In the animal model of status epilepticus, penetrate fiber stimulate (perforant path stimulation, PPS) before or after, the galanin of door week injection can shorten the persistent period (Mazaratiet al., 1998) of epilepsy.Above-mentioned influence can be reversed by applying the galanin antagonist jointly.As shown in Figure 2, the dosage that is low to moderate the 50-500 picomole just can effectively stop the existing epilepsy state of keeping certainly (SSSE).

A kind of method that is used for the treatment of epilepsy is to use the medicine based on neuropeptide.As a kind of Proof of Concept, prove that recently two kinds of non-peptide galanin receptors agonist---galnon and galmic---have convulsion and antiepileptic activity (being respectively (Saar et al., 2002) and (Bartfai et al., 2004)).Micromole's affinity that as if two kinds of chemical compounds all have moderate to GalR1 or GalR2 receptor, and their are understood show anti-convulsant activity in animal epileptic models when with its whole body administration.As described in Figure 3, galnon (2mg/kg, i.p.) or galanin (0.5nm i.c.v.) induces the incubation period of test and outbreak scoring that suitable influence is all arranged to the pentylenetetrazole (PTZ) that carries out in mice.

Following table shows NAX 5055 (GAL-BBB2) to the galanin receptors affinity.

Table 2:

Part	GalR1	GalR2
			Gal(1-16)	8.5nM	8.3nM
NAX
	5055	～9nM	～6nM
Galmic				34,000nM	>100,000nM
	Galnon	12,000nM	24,000nM

When the peritoneal injection galanin, find that it can reduce the order of severity of the epilepsy that pentylenetetrazole brings out in the mice body and the incubation period of increase epilepsy.Injection galnon also is proved to be the persistent period that can shorten from keeping the epilepsy state in the Hippocampus.Similarly, when injection in the Hippocampus or during peritoneal injection, galmic status epilepticus capable of blocking.Therefore, above-mentioned two kinds of galanin agonist are useful anticonvulsants, and have confirmed that galanin receptors can be used as the treatment of epilepsy target.

Galanin is a kind of 30 amino acid whose neuropeptides, but SAR discovers with full-length peptide and compare that N-terminal partly remains very effective agonist (Langel and Bartfai, 1998).Galanin (1-16) analog can be used in the method disclosed herein, and is wherein as follows, Gly ¹Residue by N-methyl-Gly (sarcosine, SAR) replace:

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro His Ala Val

Gly ¹The methylate N-terminal that makes described peptide of N exempt from the proteolytic degradation of acceleration, and don't can obviously change its affinity (Rivera Baeza et al., 1994) to galanin receptors.SAR discovers that following residue is most important for biological activity: Gly ¹, Trp ², Asn ⁵, Tyr ⁹And Gly ¹²(Landet al., 1991).Samely discover that the N-terminal extension can cause bioactive forfeiture.On the other hand, when the C-terminal of galanin (1-16) partly is connected on the bigger structure, its as if can become highly stable (robust) (Pooga et al., 1998).Therefore, when only considered amino acid is replaced, [Sar ¹] method for designing of galanin analog is similar to the method that is used for somatostatin, but this method is introduced at C-terminal rather than N-terminal and is extended.

When peritoneal injection, galanin analog---GAL-BBB2 (SEQ ID NO:3)---demonstrates effective anti-convulsant activity (ED50 is about 3mg/kg) (embodiment 2).Can obtain to have the galanin analog of the minimum of the most effective and long lasting anti-convulsant activity by GAL-BBB2.The example of these sequences can find (describing in detail among the embodiment 2) in SEQ ID NO:4-29.For example when comparing with galanin, these peptides have the stability of increase.The invention also discloses galanin analog GAL-BBB3, GAL-BBB4, GAL-BBB5, GAL-BBB6, GAL-BBB7 and GAL-BBB8 (SEQ IDNO:49-54).Every kind of above-mentioned analog also all has anti-convulsant activity.

Carry out limited structure-functional relationship research and differentiate the minimum GAL-BBB2 analog fragment that when being given, can keep anti-convulsant activity by whole body.Synthetic and test comprises the galanin analog of C-terminal and central truncate.In addition, the C-terminal motif is carried out limited structure-functional relationship research to optimize the blood-brain barrier permeability of described analog.Figure 14 shows the GAL-BBB2 structure in structure-functional study.

The method (referring to embodiment 1 and 2) of various galanin analog hereinafter being discussed and being used to design and synthesize them.

C) somatostatin

Exist many evidence proof brain somatostatins to play important function (Vezzani and Hoyer, 1999) as epilepsy and epilepsy invasion inhibitor.Somatostatin is a kind of main neuropeptide of expressing in the Hippocampus GABA energy relay cell.In addition, but glutamic acid stimulation in rats hippocampal neuron discharges somatostatin (Fontana et al., 1996).Behind the epilepsy, somatostatin and receptor expression generation significant change thereof, and supposed that this neuropeptide can regulate and control neuronic irritability ((Schwarzer et al. in the epilepsy invasion process, 1996) and in (Vezzani andHoyer, 1999) this is summarized).Receptor subtype knocks out in the neurotransmission with the verified glutamic acid mediation in Hippocampus of pharmacological research and relates at least four kinds of the somatostatin receptor hypotypes (sst1, sst2, sst3 and sst4) (Pikwo et al., 1996).Csaba and colleague's thereof recent research (Csaba et al., 2004) provides extra evidence, and promptly somatostatin sst2A receptor can play a key effect in epilepsy invasion and anti-convulsant activity.

The positive evidence of the anti-convulsant activity of somatostatin come from research its in the animal epilepsy model to pharmacological action (Vezzani et al., 1991 of epilepsy and epilepsy invasion; Perez etal., 1995; Mazarati and Wasterlain, 2002) (Mazarati and Wasterlain, 2002).Injection somatostatin or its subtype-selective analog cause the epilepsy number of times to reduce, and increase the incubation period (Vezzani et al., 1991) of the epilepsy that kainic acid or quinolinic acid bring out.Similarly, inject the quantity (Perez et al., 1995) that RC-160 (somatostatin sst2-selective agonist) can reduce the animal that suffers from tetanic-clonic epilepsy outbreak that pentylenetetrazole brings out.As shown in Figure 4, (i.h.) injection somatostatin can significantly reduce from the epilepsy distribution of keeping in the epilepsy state rat model (Mazarati and Wasterlain, 2002) in the Hippocampus.

Somatostatin is a kind of 14 amino acid whose hypothalamus peptides that contain a single disulfide bond, is found in 1973 at first (Brazeau et al., 1973).The sequence of somatostatin is as described below:

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Ala Gly Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys

SAR research has widely had been found that 5 Key residues: Phe ⁶, Phe ⁷, Trp ⁸, Lys ⁹And Phe ¹¹, and use alanine displacement Gly ², Lys ⁴, Asn ⁵, Thr ¹⁰, Thr ¹²Or Ser ¹³Can obviously not influence biological activity (Vale et al., 1975).In addition, proved and contained D-Trp ⁸Analog more effective because its Proteolytic enzyme resistance is stronger and/or activity conformation is more stable.

[D-Trp ⁸] or [L-Trp ⁸] somatostatin can be used as the analog of the metabolic stability that is used for method disclosed herein.In order to increase alkalescence, but Thr, Ser or Asn residue can be by similar positively charged DAB of isosterism (DAB) or the systematically displacements of DAP (diaminopropionic acid) residue institute.For increasing lipotropy, can introduce the Lys-palmityl and partly replace Lys ⁴Or Asn ⁵, and/or use the halogenation equivalent chloro-Phe residue of Phe residue to replace the Phe residue.As summing up in the table 5, synthesized nine kinds of analog and measured them and the affinity of the somatostatin receptor.With combination does not cause the modification of negative influence to combine to high-affinity.Above-mentioned second filial generation analog contains the modification of 2-4 kind combination.These sequences can find in SEQ ID NO:31-36.

Next, the N-terminal extension is incorporated into [D-Trp ⁸] or [L-Trp ⁸] in the somatostatin.Above-mentioned extension (as shown in Figure 8, BBB/PK is modified) have a dual purpose: (1) utilizes passive and active mechanisms to improve blood-brain barrier permeability and (2) by adding the pharmacokinetic property that the huge part (bulky moiety) that can reduce clearance rate and improve the proteolytic degradation resistance is improved the neuropeptide medicine.Because this class " BBB/PK is modified " is a new notion, therefore, use several combinations of the construction unit of rare several formation extensions.Table 6 provides the unitary 26S Proteasome Structure and Function information about being recommended.

The invention also discloses the octreotide that can be used for compositions disclosed herein and method.For example, SEQID NO:40 discloses a kind of octreotide molecule.Figure 21 shows chemical constitution and the sketch map of transforming octreotide.Octreotide is a kind of somatostatin analogs responsive more to the sst2 hypotype of the somatostatin receptor (having 5 kinds of known hypotypes).Proved in epilepsy and the epilepsy invasion and related to somatostatin.Following list of references is included this description in about the whole modes by reference of the instruction of octreotide, somatostatin and epilepsy: Vezzani A and Hoyer D, and Eur J Neurosci, 1999, vol 11, pp3767-3776.Similarly, Chapman V and Dicjkenson AH, Neuropept ides 1992, vol 23, proved the effect of somatostatin in nociception among the 147-152, this list of references is included this description in about the whole modes by reference of the instruction of somatostatin, octreotide and nociception.Somatostatin has been described recently at the developing effect of Alzheimer (Saito T et al, Nature Medicine, 2005, vol 11, p.434-439, this list of references is included this description in about the whole modes by reference of the instruction of octreotide, somatostatin and Alzheimer.Hereinafter discussed and be used to design and the method (referring to embodiment 1) of synthetic somatostatin analogs.

D) δ-sleep inducing peptide

δ-sleep inducing peptide (DSDP) is that (Schoenenberger 1984 for a kind of convulsion neuropeptide; Kovalzon and Strekalova 2006).DSIP and dermorphin (a kind of [mu agonist) have some structural similarities.DSIP can effectively suppress epilepsy in the epilepsy model that metaphit brings out.In addition, proved that DSIP can strengthen the anti-convulsant activity of valproate (Hrnc ic, Stanojlovic et al.2006) in identical epilepsy model.In addition, prove the interaction between this peptide scalable enkephalin and the Opioid Receptors, thereby made DSIP have analgesic activity (Nakamura, Nakashima et al.1986).In a kind of toxicity cerebral edema model, proved the neuroprotective activity of DSIP.It is reported DSIP and the penetrable BBB of some analog (Kastin, Nissen etal.1981; Kastin, Banks et al.1982).

2. the compositions that has enhanced blood-brain barrier permeability

Verified compositions disclosed herein has enhanced blood-brain barrier permeability.As described herein, the invention discloses the set of neuropeptide analog, designed and synthesized described neuropeptide analog to test them with the ability of high-affinity in conjunction with its each autoreceptor.Every kind of neuropeptide comprises about 10 kinds of analog in this set.Further test high-affinity analog penetrates the ability of blood brain barrier.Obtain the result of first generation analog, synthetic then and assessment second filial generation analog is followed by third generation analog.Select most promising analog (high-affinity part) to confirm their agonist activity by functional examination with enhanced blood-brain barrier permeability.In vivo a subclass (the effective agonist with enhanced blood-brain barrier permeability) of above-mentioned analog is carried out pharmacology's test then.

For becoming medicine, the neuropeptide analog should have several important properties, comprising: (1) is high tires and the half-life that selectivity, (2) metabolic stability, (3) are long relatively and the body loop cleaning rate of reduction, and (4) enhanced blood-brain barrier permeability.Most of neuropeptides demonstrate high tiring and selectivity.Metabolic stability can not introduced by the residue displacement susceptible residue that proteolytic enzyme is discerned by peptide backbone modifications and/or use usually.Can realize the increase of half-life and the reduction of eliminating speed effectively by a part based on polymer is puted together (for example, Pegylation) with peptide.Higher blood-brain barrier permeability can pass through to increase lipotropy or cationization, and introduces by adding prodrug, nutrition transhipment analogies or glycosylation.The structure of ideal medicine neuropeptide illustrates in Fig. 8 schematically.

As shown in Figure 8, new ideas in the neuropeptide engineering are " BBB/PK are modified ".Described BBB/PK is modified comprise have lipophilic, cation and the unitary huge part of transhipment analogies based on polymer; This is modified to have dual purpose: strengthen blood-brain barrier permeability and improve pharmacokinetic property.Described cation and lipophilic unit can promote respectively and negative electricity film surface between interaction and raising stride film diffusion.The function of described active transport model configuration is to improve the specificity of brain capture neuropeptide by enhancing and the interaction between the lip-deep specificity nutrition of brain endothelial cell transport protein.The structural framing that contains whole said units can also improve the pharmacokinetic property of described peptide, the effect of the peg moiety that simulation/replacement is commonly used.Test is as the N of described model neuropeptide or the above-mentioned huge part of C-terminal extension, and this paper also discloses more general link position in the described neuropeptide structure.

Use following method design to have the neuropeptide analog of enhanced blood-brain barrier permeability: if possible, to use the analog of metabolic stability to begin.Differentiate and to carry out metathetical other AA positions of side chain in the described analog.Discriminating is positioned at the position that N and C-terminal can be introduced huge part.Replace lipotropy and the alkalescence that increases analog by side chain.Introduce extension to peptide analogues, described extension will further increase its lipotropy and alkalescence, improves pharmacokinetic property (BBB/PK is modified) simultaneously.Comprise that at described extended spot a nutrition model configuration is to improve the specificity that blood brain barrier penetrates.Make up described analog and described extension (BBB/PK is modified) that side chain is modified that have.

The key that success designs this class analog is the correct combination of above-mentioned modification.For realizing this goal, when each set that designs and assess modification and their best of breed, can adopt system approach.Group method illustrates in Figure 10 schematically.Disclosed hereinly can in the solid-phase peptide building-up process, use the automatic peptide synthesizer to introduce to amino acid whose modification.All alpha-non-natural amino acids or put together the derivant that structure all is commercially available Fmoc protections.

It should be understood that, when mentioning variant, particular permutation shown in the described variant basis has special properties, yet, also considered other displacements, disappearance and/insert (for example in non-position preservative replacement, insertion and/or the disappearance of indicating the position especially), prerequisite is that described variant has kept disclosed activity.

The invention discloses the have required character galanin analog of (for example enhanced blood-brain barrier permeability).The invention also discloses somatostatin analogs with enhanced blood-brain barrier permeability.As hereinbefore defined, " increase " or " enhanced " be meant that the described compositions of higher proportion can be passed blood brain barrier with wild type, that do not transform or native peptides is compared or compare with reference composition.For example, with the contrast, peptide or compositions natural or wild type are compared, and the ratio of increase can be percent 1, and 2,3,4,5,6,7,8,9,10,11,12,13,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,125,150,175,200,225,250,275,300,325,350,375,400,425,450,475,500,550,600,650,700,750,800,850,900,950,1000,1250,1500,1750,2000,2250,2500,2750,3000,3500, or 4000.

It should also be understood that every kind of analog individuality being discussed in the embodiment form also has basic permeability, described basic permeability can be determined by disclosed compositions is active.It should be understood that except as otherwise noted the above-mentioned active percent that increases can calculate by wild type, the basic permeability natural or control peptide that random time obtains, described basic permeability can provide the data in the described determination and analysis scope.

As disclosed in embodiment 1 and 2, the invention discloses displacement, disappearance, modification, addition and extension that the peptide to known or wild type carries out.For example, disclosing the N-terminal that somatostatin is carried out in the table 7 extends.Extension disclosed herein can be used for natural, wild type or known peptide, perhaps can be in conjunction with the analog that is used for known peptide.For example, can carry out side chain to known peptide and modify, combine with extension disclosed herein then.

The invention also discloses amino acid replacement and addition, what wherein said displacement or addition were used is the material that a kind of non-natural exists.Example includes but not limited to sarcosine, DAB (DAB), diaminopropionic acid (DAP), Lys-palmityl, chlorophenylalanine (Chloro-phe), aminocaproic acid (AHX), perfluor caproic acid (PerFHX), 8-amino-3, the sad and few lysines of 6-two oxa-s (oligo-Lys), Terleu.

The invention also discloses use " main chain prosthese (backbone prothesesis) " (for example non-peptide sept) replacement amino acid residue.Example includes but not limited to aminovaleric acid or aminocaproic acid.This can make whole molecular dimension minimum and don't obviously change interval between the Key residues.For example, described sept can be replaced 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,30,40 or 50 residue.

The invention also discloses amino acid whose version, wherein amino acid whose conformation changes.For example, the invention discloses D-Lys, D-Trp and L-Trp.

The invention discloses the analog of known compound (for example galanin and somatostatin), for example have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% homogeneity, and wherein said analog comprises at least 1, at least 2, at least 3, at least 4, at least 5 or at least 6 any displacements disclosed herein, disappearance, addition or extensions with parental generation sequence (for example galanin or somatostatin).

3. sequence similarity

It should be understood that as discussed herein used term homology is identical with the similarity implication with homogeneity.Therefore, for example, if the term homology be used for then it should be understood that the evolutionary relationship that needn't illustrate between the above-mentioned two sequences between two non-natural sequences, but should be conceived to similarity or sibship between its nucleotide sequence.Many methods that are used for homology between definite two evolution correlation molecules are applicable to any two or more nucleic acid molecules or albumen usually so that measure sequence similarity, and do not consider whether they are to evolve to be correlated with.

Usually, it should be understood that a kind of method that defines gene disclosed herein or proteic any known variant and derivant or variant that may occur and derivant is to define described variant or derivant by the homology with specific known array.This homogeneity of particular sequence disclosed herein is also discussed to some extent in other places of this paper.Generally speaking, gene disclosed herein and proteic variant usually have homology at least about percent 40,50,55,60,65,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98 or 99 with described sequence or native sequences.Those skilled in the art can easily understand homology how to determine two kinds of albumen or nucleic acid (for example gene).For example, can after being compared, two sequences calculate homology so that described homology can be in its top level.

The another kind of method of calculating homology can utilize disclosed algorithm to implement.The sequence alignment that can be used for comparison: local homology's algorithm of Smith and Waterman Adv.Appl.Math.2:482 (1981) by following method optimization, Needleman and Wunsch, the homology alignment algorithm of J.MoL Biol48:443 (1970), Pearson and Lipman, the similarity method research of Proc.Nat l.Acad.Sci.U.S.A.85:2444 (1988), the computer of above-mentioned algorithm is carried out (GAP, BESTFIT, FASTA, and TFASTA in the Wiscons in Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI) or the check.

For example can pass through Zuker, M.Science 244:48-52,1989, Jaeger et al.Proc.Natl.Acad.Sci.USA 86:7706-7710,1989, Jaeger et al.Methods Enzymol183:281-306,1989 disclosed algorithms obtain the nucleic acid homology of same type, and data mode by reference relevant with the nucleic acid comparison at least in the described list of references is included this description in.It should be understood that, usually can use any method and the possibility of result of above-mentioned the whole bag of tricks can be different in some cases, use at least a said method to find homogeneity but the technical staff will appreciate that, then described sequence should be called as and has described homogeneity and by disclosed herein.

For example, as used herein, the sequence that is described to have with another sequence certain particular percentile homology is meant the sequence with described homology, and described homology is to utilize one or more computational methods mentioned above to calculate.For example, as herein defined, has 80% homology if use the computational methods of Zuker to calculate first sequence and second sequence, do not have 80% homology even then utilize other any computational methods to calculate described first sequence and second sequence, described first sequence and second sequence also have 80% homology.As another example, as herein defined, has 80% homology if use the computational methods of the computational methods of Zuker and Pearson and Lipman to calculate first sequence and second sequence, do not have 80% homology even then utilize the computational methods of computational methods, Jaeger of computational methods, the Needleman and Wunsch of Smith and Waterman or other any computational methods to calculate described first sequence and second sequence, described first sequence and second sequence also have 80% homology.As another example, as herein defined, have 80% homology (although in fact different computational methods will draw different calculating homology percent usually) if use every kind of computational methods all to calculate first sequence and second sequence, then described first sequence and second sequence have 80% homology.

4. nucleic acid

There is multiple peptide molecule disclosed herein, for example various galanins and somatostatin analogs.It should be understood that, molecule based on above-mentioned peptide can be coded by many nucleic acid, comprise for example nucleic acid of SEQ ID NO:1-55 of for example encoding, for example it should be understood that the mRNA that is expressed will be made up of A, C, G and U usually when carrier during at cell inner expression.

A) sequence

Have many sequences relevant with for example galanin, described sequence can for example find among the Genbank data base, and described Genbank data base can land the www.pubmed.gov visit.Above-mentioned sequence and other sequences and the wherein contained whole modes by reference of each sequence are included this description in.

This paper uses a concrete sequence of listing among the SEQ ID NO:3 to illustrate disclosed compositions and method as an example.It should be understood that unless otherwise specified the description relevant with this sequence is applicable to any any sequence relevant with the galanin analog.How to it will be understood by those skilled in the art that analytical sequence deviation and difference and how to adjust the compositions relevant with particular sequence and method to be applicable to other correlated serieses (being galanin analog sequence).For example, consider information disclosed herein and known in the art, can be at galanin relevant nucleotide sequence design primer and/or probe.

5. compositions is delivered to (carrier) in the cell

Exist many can being used in external or body, nucleic acid or delivery of peptides to be arrived intracellular compositions and method.Carrier disclosed herein can be used for many aspects.In an example, carrier disclosed herein can be used for coding delivery of peptides disclosed herein to cell and experimenter.As indicated above, carrier can also be used to peptide and pass blood brain barrier to help it.

The method and composition relevant with carrier can be divided into two classes substantially: based on the delivery system of virus with not based on the delivery system of virus.For example, nucleic acid and peptide can be sent by many direct delivery systems, for example electroporation, fat transfection, calcium phosphate precipitation, plasmid, viral vector, viral nucleic acid, bacteriophage nucleic acid, phage, cosmid; Perhaps by transfer of genetic material is for example sent in the cationic-liposome to cell or carrier.Wolff for example, J.A., et al., Science, 247,1465-1468, (1990); And Wolff, J.A.Nature, 352,815-818, (1991) have been described the transfection method that is fit to and have been comprised viral vector, chemical transfection or physico-chemical process, for example electroporation and dna direct diffusion.These class methods are well known in the art, and are easy to be carried out modification to be applicable to compositions as herein described and method.In some cases, described method will be modified to be applicable to big dna molecular specifically.By using the targeting feature of described carrier, said method can also be used for some disease of targeting and cell mass.Send for the blood brain barrier of striding that further improves described compositions, can use the TAT nexin transduction domain (Dietz GP and Bahr M, Mol Cell Neurosci, 2004, vol 27, p.85-131).

Plasmid used herein or viral vector are can be with in disclosed nucleic acid or peptide (for example those nucleic acid or peptides relevant with galanin and the somatostatin analogs) transporte to cells and the material of not degrading.In some embodiments, described delivery system is derived from virus or retrovirus retrovirus.Viral vector is, for example, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poliovirus, AIDS virus, neuron nutrition virus (neuronal trophic virus), Sindbis and other RNA viruses comprise the virus with HIV skeleton.Also preferred any and above-mentioned virus is shared the virus family of some character, and described character makes above-mentioned virus be suitable as carrier.The retrovirus retrovirus of required character when retrovirus retrovirus comprises Mus Moloney Leukemia virus, MMLV and can express MMLV as carrier.Retrovirus retrovirus can carry a hereditary load bigger than other viral vector, i.e. transgenic or marker gene, and be a kind of carrier commonly used therefore.Yet they can not be used for nonproliferating cell.Relatively stable and the easy use of adenovirus vector has that height is tired and can send with the aerosol preparations form, and can the transfection Unseparated Cell.The Pox viral vector is big and have the site that several are used to insert gene, and they are heat-staple and can at room temperature preserve.Embodiment preferred is the viral vector of process transformation with the immunne response of inhibition host living beings, and described immunne response is caused by described virus antigen.Preferred this class carrier will carry the coding region of interleukin 8 or 10.

Viral vector can have than gene being introduced the higher transfection ability of intracellular chemistry or physical method.Usually, viral vector contains non-structure early gene, structure late gene, rna plymerase iii transcript, duplicates and pack necessary reverse terminal repeat, and is used to regulate and control the promoter that described viral genome is transcribed and duplicated.When being transformed into carrier, virus has one or more early genes to be removed usually, and gene or gene/promoter box are inserted into the viral DNA that replacement is removed in the described viral genome.This class construct can carry the exogenous genetic material that reaches about 8kb most.The essential function of the early gene that is removed is provided by cell line usually, described cell line be transformed into can the described early gene of trans expression gene outcome.

(1) retrovirus vector

Retrovirus retrovirus is a kind of animal virus that belongs to Retroviridae, comprises any kind, subfamily, genus or tropism (tropism).Verma, I.M., Retroviral vectors for gene transfer.In Microbiology-1985, American Society for Microbiology, pp.229-232, Washington has described common retrovirus vector in (1985), described list of references mode is by reference included this description in.Use example that retrovirus vector carries out gene therapy at United States Patent (USP) 4,868,116 and 4,980,286; PCT application WO 90/02806 and WO 89/07136; And describe to some extent among the Mulligan (Science 260:926-932 (1993)), the instruction of described list of references mode is by reference included this description in.

Retrovirus retrovirus is exactly a kind of nucleic acid load to be wrapped into wherein packing basically.Described nucleic acid load carries packaging signal, and described packaging signal can guarantee that the daughter DNA that is replicated is packaged in the described Package casing effectively.Except that packaging signal, also there are many described required cis molecules of virus that are replicated that duplicate and pack.The retrovirus retrovirus genome comprises and relevant gag, pol and the env gene of the described protein coat of preparation usually.The foreign DNA that described gag, pol and env gene can will be transferred in the target cell is usually replaced.Retrovirus vector contain usually be useful on the sequence that merges to the described gag transcriptional units of packaging signal, the signaling transcriptional start in the described Package casing, carry out the required element of reverse transcription comprise the primer binding site that is used in conjunction with the tRNA primer of reverse transcription, at the terminal repeat of DNA building-up process guide RNA chain conversion, as 5 ' to 3 ' the LTR sequence that is rich in purine of the synthetic initiation site of second DNA chain, and near the particular sequence the LTR end, described LTR can make the retrovirus retrovirus of DNA attitude be inserted in the host genome.Removing of gag, pol and env gene make and the exogenous array of about 8kb can be inserted in the described viral genome, become reverse transcription and be wrapped into when duplicating in the new counter-transcription-ing virus particle.The amount of nucleic acid is enough to send one to a plurality of genes, depends on the size of every kind of transcript.Preferably, comprise positive or negative selected marker and other genes in the described insert.

Because replicanism and packaging protein in most of retrovirus vectors are removed (gag, pol and env), so produce described carrier by described carrier is put into package cell line usually.Package cell line is the cell line that has been inverted the record virus transfection or had transformed, and described retrovirus retrovirus contains the mechanism of duplicating and pack and still do not contain any packaging signal.Transfected in the above-mentioned cell line time when the carrier that carries selected DNA, the cis mechanism of utilizing accessory cell to provide is duplicated the carrier that contains genes of interest and it is packaged in the new counter-transcription-ing virus particle.The genome that is used for described mechanism is not packaged, because they lack the packing desired signal.

(2) adenovirus vector

Following list of references is described the structure of replication-defective adenoviral

(Berkner et al.，J.Virology 61：1213-1220(1987)；Massie et al.，Mol.Cell.Biol.6：2872-2883(1986)；Haj-Ahmad et al.，J.Virology 57：267-274(1986)；Davidson et al.，J.Virology61：1226-1239(1987)；Zhang＂Generation and identification of recombinant adenovirus byliposome-mediated transfection and PCR analysis＂BioTechniques 15：868-872(1993))。

Use these viruses to be that as the benefit of carrier the spread scope that they propagate in other cell types is restricted, because they can duplicate in initial infected cells but can not form new infectious virus particle.Proved recombinant adenovirus directly has been delivered to after airway epithelia, hepatocyte, blood vessel endothelium, CNS essence and many other tissue sites in vivo, they can realize gene transfer efficiently (Morsy, J.Clin.Invest.92:1580-1586 (1993); Kirshenbaum, J.Clin.Invest.92:381-387 (1993); Roessler, J.Clin.Invest.92:1085-1092 (1993); Moullier, NatureGenetics 4:154-159 (1993); La Salle, Science 259:988-990 (1993); Gomez-Foix, J.Biol.Chem.267:25129-25134 (1992); Rich, Human Gene Therapy 4:461-476 (1993); Zabner, Nature Genetics 6:75-83 (1994); Guzman, Circulation Research 73:1201-1207 (1993); Bout, Human Gene Therapy 5:3-10 (1994); Zabner, Cell 75:207-216 (1993); Caillaud, Eur.J.Neuroscience 5:1287-1291 (1993); And Ragot, J.Gen.Virology 74:501-507 (1993)).

Recombinant adenovirus is by realizing gene transfer in conjunction with the specific cells surface receptor, afterwards should virus by receptor-mediated endocytosis institute internalization, mode is identical with wild type or replication-defective adenoviral

(Chardonnet and Dales，Virology 40：462-477(1970)；Brown and Burlingham，J.Virology12：386-396(1973)；Svensson and Persson，J.Virology 55：442-449(1985)；Seth，et al.，J.Virol.51：650-655(1984)；Seth，et al.，Mol.Cell.Biol.4：1528-1533(1984)；Varga et al.，J.Virology 65：6061-6070(1991)；Wickham et al.，Cell 73：309-319(1993))。

Viral vector can be based on the virus of the adenovirus that has removed the E1 gene, and above-mentioned virion can produce in the cell line of for example people's 293 cell lines.In another preferred embodiment, E1 and E3 gene all are removed from the adenoviral gene group.

(3) gland relevant viral vector

Another kind of viral vector is based on adeno-associated virus (AAV).This deficiency parvovirus is preferred carrier because it can infect many cell types and concerning the people right and wrong pathogenic.AAV class carrier can be transported about 4-5kb, and known wild type AAV can stably be inserted in the chromosome 19.The carrier that preferably has this site-specific integration characteristic.A particularly preferred embodiment of this class carrier is the P4.1C carrier of being produced by the Avigen that is positioned at San Francisco, described carrier contains herpes simplex virus thymidine kinase gene, HSV-tk and/or marker gene, for example the gene of encoding green fluorescent protein (GFP).

In another kind of AAV virus, described AAV contains the reverse terminal repeat (ITR) that a pair of its flank is connected at least one expression cassette, and described expression cassette contains a promoter that instructs cell specific expression that effectively is connected with heterologous gene.Allos is meant that any is the nucleotide sequence or the gene of non-natural concerning AAV or B19 parvovirus herein.

Usually, described AAV and B19 coding region are lacked, and produce a kind of carrier of safe no cytotoxicity.Described AAV ITR or its modified forms can be given infectivity and site-specific integration and non-cell toxicity, and described promoter can instruct cell specific expression.United States Patent (USP) 6,261, data by reference the mode relevant with the AAV carrier included this description in 834.

Therefore, carrier of the present invention provides and can be incorporated in the mammal chromosome and nontoxic basically dna molecular.

The gene that is inserted in virus and the retrovirus retrovirus contains helpful regulation and control genes of interest product expression promoter and/or enhancer usually.Promoter generally be one or more when its be in the relatively-stationary position of transcriptional start site on the time can bring into play the DNA sequence of function.Promoter contains carries out the required core parts of basic interaction between RNA polymerase and the transcription factor, and may contain upstream element and response element.

(4) heavy load viral vector

The molecular genetic experiment of carrying out with large-scale herpes virus hominis provides a kind of method, rely on this method can in the cell of available herpesvirus infection, clone, increase and place big allogeneic dna sequence DNA fragment (Sun et al., Nature genetics 8:33-41,1994; CotterandRobertson .Curr Opin Mol Ther 5:633-644,1999).Above-mentioned large-scale DNA viruses (herpes simplex virus (HSV) and epstein-Barr virus (EBV)) can be delivered to the people's allogeneic dna sequence DNA fragment greater than 150kb in the specific cell.The EBV recombinant can keep big dna fragmentation as dissociative DNA in the infected B cell.Each that carries the people's gene group insert that is up to 330kb is cloned in and seems stable in the heredity.Keeping these episomes needs a kind of specific EBV nucleoprotein, and EBNA1, described nucleoprotein are constitutive expressions in the process that infects with EBV.In addition, above-mentioned carrier can be used to transfection, and can produce a large amount of albumen momently this moment external.Herpesvirus amplicon system also is used to pack greater than the dna fragmentation of 220kb and is used for infecting and can stably keeps DNA and be used as episomal cell.

Other useful systems comprise, for example duplicate the non-bean seedlings viral vector that duplicates with host's restriction.

B) not based on the system (non-nucleic acid based system) of nucleic acid

Can in many ways disclosed compositions (nucleic acid of the galanin analog of for example encoding) be delivered in the target cell.For example, can send described compositions by electroporation or by the fat transfection or by calcium phosphate precipitation.The selection of delivery mechanism will depend in part on the targeting of wanting cell type and described send for example take place in vivo or ectogenetic.

Therefore, described compositions can comprise---except that disclosed variant or carrier---and for example lipid such as liposome, for example cationic-liposome (for example DOTMA, DOPE, DC-cholesterol) or anionic liposome.If necessary, liposome can also include the albumen that helps the targeting specific cells.The compositions that contains chemical compound and cationic-liposome can be administered in the blood that can flow into target organ or be drawn in the respiratory tract that leads to the respiratory tract target cell.About liposome, see also Brigham et al.Am.J.Resp.Cell.Mol.Biol.1:95-100 (1989); Felgner et al.Proc.Natl.Acad.Sci USA 84:7413-7417 (1987); U.S.Pat No.4,897,355.In addition, the microcapsule component that described chemical compound can be used as targeting particular cell types (for example macrophage) is given, and perhaps wherein said chemical compound is from the diffusion of described microcapsule or send and be designed to realize special speed or dosage.

Foreign DNA is given and absorb in the method for (being gene transfer or transfection) in experimenter's the cell mentioned above, can described compositions be delivered in the cell via various mechanism.As an example, can send via liposome, use for example LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc. of commercially available liposome product, Gaithersburg, MD), SUPERFECT (Qiagen, Inc.Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, WI), and other are according to the liposome of the preparation of the standard step in this area.In addition, can utilize electroporation that nucleic acid of the present invention or carrier are delivered in the body, the technology that is used to carry out electroporation can be available from Genetronics, Inc. (San Diego, CA) and utilize the SONOPORATION machine (ImaRxPharmaceutical Corp., Tucson, AZ).

Described material can form suspension (for example including in microparticle, liposome or the cell) in solution.Above-mentioned substance can come the specific cell type of targeting by antibody, receptor or receptors ligand.Following list of references is to use this technology to make the example of specific protein target tumor tissue

(Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br.J.Cancer, 60:275-281, (1989); Bagshawe, et al., Br.J.Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol.Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog.Reviews, 129:57-80, (1992); And Roffler, et al., Biochem.Pharmacol, 42:2062-2065, (1991)).Above-mentioned technology can be used for various other specific cell types.Carrier for example the liposome (lipid that comprises mediation drug targeting colon carcinoma) puted together of " stealth " and other antibody, the receptor by cell specific ligand mediated dna targeting, instruct the lymphocyte of cancer target, and the treatment retrovirus retrovirus of the high degree of specificity of targeted mouse glioma cell in vivo.Following list of references be to use this technology make specific protein target tumor tissue example (Hughes et al., Cancer Research, 49:6214-6220, (1989); With Litzingerand Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)).Generally speaking, the endocytosis approach can relate to receptor (composing type or part induction type).Above-mentioned receptor clustering enters cell by clathrin-coated vesicle in clathrin-coated pit, pass describedly to be known from experience therein by the acid endosome of sorting, be re-circulated to then cell surface be stored in the born of the same parents in or in lysosome, be degraded.The internalization approach can be brought into play various functions, for example nutrition intake, the conditionality of the removing of activated protein, macromolecular removing, virus and toxin enter, the dissociating and degrading of part, and the adjusting of acceptor levels.Many receptors are followed approach in a plurality of born of the same parents, depend on cell type, acceptor density, part type, part atomicity and ligand concentration.The molecule of receptor-mediated endocytosis and cell mechanism in following list of references, obtained the summary (Brown and Greene, DNA and Cell Biology10:6,399-409 (1991)).

Be delivered to intracellular nucleic acid and contain integration sequence usually, described nucleic acid will be integrated in the host cell gene group.The normally viral correlated series of above-mentioned sequence is when particularly using viral based system (viral based systme).Above-mentioned viral integrase system can also be incorporated into and will use not in the nucleic acid of sending based on the delivery system (non-nucleic acid based system) (for example liposome) of nucleic acid, so that contained nucleic acid can be incorporated in the host genome in the described delivery system.

Other routine techniquess that are used for being incorporated into host genome comprise, for example, are designed for the system that promotes with the host genome homologous recombination.Said system depends on the sequence that flank is connected to nucleic acid to be expressed usually, target sequence in described sequence and the host cell gene group has enough homologys, vector nucleic acid and target nucleic acid are recombinated in described host cell gene group, and the nucleic acid that causes being sent is incorporated in the host gene.The necessary system and method for above-mentioned promotion homologous recombination is that those skilled in the art are known.

C) in the body/in vitro

As indicated above, described compositions can be come administration and can be in vivo and/or in vitro utilize various mechanism as known in the art (for example absorb naked DNA, liposome and merge, utilize particle gun intramuscular injection DNA, endocytosis etc.) that it is delivered in experimenter's cell with pharmaceutically suitable carrier form.

If adopt in vitro method, then can emigrated cells or tissue and carry out In vitro culture according to standard scheme as known in the art.Can described compositions be incorporated in the cell by arbitrary gene transfer mechanism, for example gene delivery, electroporation, microinjection or the proteoliposome of calcium phosphate mediation.Then, the cell that can will be transduceed according to the standard method that is applicable to this cell or tissue type injects (for example with pharmaceutically suitable carrier form) or homotopically transplants back in the described subject.Become known for various cell transplantations or the intravital standard method of injection experimenter.

6. expression system

Be delivered to intracellular nucleic acid and contain the expression regulation system usually.For example, the gene that is inserted in virus and the retrovirus retrovirus system contains helpful regulation and control genes of interest product expression promoter and/or enhancer usually.Promoter generally be one or more when its be in the relatively-stationary position of transcriptional start site on the time can bring into play the DNA sequence of function.Promoter contains carries out the required core parts of basic interaction between RNA polymerase and the transcription factor, and may contain upstream element and response element.

A) viral promotors and enhancer

The preferred promoter that the regulation and control carrier is transcribed in mammalian host cell can be available from different sources, viral genome for example is as: polyoma, simian virus 40 (SV40), adenovirus, retrovirus retrovirus, hepatitis B virus and cytomegalovirus most preferably; Perhaps can be available from the allos mammalian promoter, β actin promoter for example.Can obtain early stage and late promoter easily as the SV40 virus of SV40 restriction fragment, described restriction fragment also contain SV40 virus replication starting point (Fiers et al., Nature, 273:113 (1978)).Can obtain easily as the human cytomegalic inclusion disease virus of HindIIIE restriction fragment immediate early promoter (Greenway, P.J.et al., Gene18:355-360 (1982)).Certainly, the promoter from host cell or relevant species also can be used for the present invention.

Enhancer typically refers in the DNA sequence that plays a role with the unfixed place of the distance of described transcriptional start site, and can be positioned at described transcript unit 5 ' (Laimins, L.et al., Proc. Natl.Acad.Sci.78:993 (1981)) or 3 ' end (Lusky, M.L., et al., Mol.Cell Bio.3:1108 (1983)).In addition, enhancer can be arranged in intron (Banerji, J.L.etal., Cell33:729 (1983)) and in the described coded sequence itself (Osborne, T.F., etal., Mol.Cell Bio.4:1293 (1984)).Their length is usually between 10 to 300bp, and they play a role with cis.Enhancer plays a role to be increased by initial the transcribing of promotor-proximal.Enhancer also usually contains the response element that mediates transcriptional regulatory.Promoter can also contain the response element that mediates transcriptional regulatory.Enhancer usually can determine the adjusting to gene expression.Though present known many enhancer sequence that derive from mammalian genes (globin, elastoser, albumin, fetoprotein and insulin), research worker are used the enhancer that derives from eukaryotic cell virus to carry out generality usually and are expressed.Preferred examples is for being positioned at the sub-enhancer of SV40 enhancer, cytomegalovirus early promoter of origin of replication (bp100-270) side in late period, the polyoma enhancer that is positioned at origin of replication side in late period and adenovirus enhancer.

Described promoter/enhancer can activate specifically by the specificity chemical event that light maybe can trigger its function.For example can using, the reagent of tetracycline and dexamethasone comes regulating system.Also exist by being exposed to the method that irradiation (for example γ irradiation) or alkanisation chemotherapeutic agent come the enhanced virus vector gene to express.

In certain embodiments, the effect of constitutive promoter and/or enhancer can be played so that the expression maximum in the transcript unit zone that will transcribe in described promoter and/or enhancer zone.In some construct, described promoter and/or enhancer zone all have activity in all eukaryotic cell types, although described promoter and/or enhancer zone can only be expressed in specific cell type in the specific time.A kind of preferred this class promoter is CMV promoter (650 base).Other preferred promoteres are SV40 promoter, cytomegalovirus (total length promoter) and retrovirus vector LTF.

Verified all specificity regulating elements all be can clone and can be used to make up the expression vector that can in particular cell types (for example melanoma cell), be expressed by selectivity.Glial fibrillary acidic protein (GFAP) promoter has been used to the gene in the sexual cell of selective expression's colloid source.

Used expression vector can also contain and stops transcribing required sequence in the eukaryotic host cell (yeast, fungus, insecticide, plant, animal, people or nucleated cell), and described sequence can influence the expression of mRNA.Above-mentioned zone is transcribed into the polyadenylation section in the untranslated part of mRNA of coding tissue factor protein.Described 3 ' untranslated region also comprises the tanscription termination site.Preferably, polyadenylation region is also contained in described transcript unit.A benefit in this zone be it can increase described by transcript unit with mRNA equally processed and the transhipment probability.Discriminating and the purposes of polyadenylation signal in expression vector is known.Preferably, in described transgenic constructs, use the homology polyadenylation signal.In some transcript unit, described polyadenylation region derives from the early stage polyadenylation signal of SV40 and by about 400 base compositions.Also preferably, described can be contained other standard sequences separately by transcript unit or contain other standard sequences and above-mentioned sequence so that promote the expression of described construct or increase its stability.

B) labelling

Described viral vector can comprise the nucleotide sequence of coded markings product.This marked product is used to determine whether described gene is delivered in the cell, in case and described gene sent then expression immediately.Preferred marker gene is an escherichia coli lacZ gene, described gene code beta galactosidase; And green fluorescence protein gene.

In some embodiments, described labelling can be a selection markers.The example that is suitable for the selection markers of mammalian cell is dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin and puromycin.When this class selection markers is successfully transferred in the mammalian host cell, if place the next mammalian host cell that is transformed of screening pressure to survive in cell.The different screening technique that has two class extensive uses.The first kind is based on cellular metabolism and uses mutational cell line, and described cell line lacks the ability that supplementing culture medium is independently grown that do not rely on.Two examples are CHO DHFR cell and mice LTK cell.Above-mentioned cell lacks the ability of growing under the situation of not adding such as nutrients such as thymidine and hypoxanthine.Because above-mentioned cell lacks necessary some gene of complete nucleotide route of synthesis,, they in supplementing culture medium, provide the nucleotide that is lacked unless can't surviving.A kind of alternative method of replenishing described culture medium is that complete DHFR and TK gene are introduced in the cell that lacks corresponding gene, therefore changes its growth demand.Do not use each cell of DHFR or TK gene transformation in non-supplementing culture medium, not survive.

Second class is that advantage is selected, and described selection is meant the selection scheme that can be used in any cell type and do not need to use mutational cell line.Such scheme uses medicine to hinder the growth of host cell usually.Under those cells with new gene will be expressed and can be given the drug-fast albumen of cell and will survive in screening.The example use medicine neomycin that this advantage is selected (Southern P.and Berg, P., J.Molec.Appl.Genet.1:327 (1982)), Mycophenolic Acid (Mulligan, R.C.andBerg, P. Science209:1422 (1980)) or hygromycin (Sugden, B.et al., Mol. Cell.Biol.5:410-413 (1985)).Above-mentioned three examples adopt and are subjected to the bacterial gene of eukaryotic cell regulation and control to give the resistance of cell to suitable medicine G418 or neomycin (Geneticin), xgpt (Mycophenolic Acid) or hygromycin respectively.Other medicine comprises neomycin analog G418 and puromycin.

7. peptide

A) protein variant

As described herein, there are many known and peptide variants of having considered in this article.In addition, for position disclosed and disclosed herein relevant functional variant and analog, have known functional natural variant on non-position disclosed herein, described natural variant also has required function.Protein variant and derivant are known in those skilled in the art and can contain amino acid sequence modifications or functional fragment.For example, amino acid sequence modifications can be divided into usually following three apoplexy due to endogenous wind one or more: the displacement variant, insert variant or disappearance variant.Insertion comprises that amino and/or carboxyl terminal merge, and inserts in the sequence of single or multiple amino acid residues.Insertion is generally than amino or carboxyl terminal merges little insertion, for example 1-4 residue.Immunogenicity solvent protein derivative (for example those described in the embodiment) can be by using external crosslinked will the preparation even as big as producing the reconstitution cell culture that immunogenic polypeptide and target sequence merge or transformed by the DNA that uses this fusion rotein of coding.Disappearance is characterised in that and removes one or more amino acid residues from described protein sequence.Usually, disappearance is no more than about 2-6 residue on the arbitrary site in described protein molecular.These variants prepare according to following step usually: carry out the direct mutagenesis of nucleotide in the DNA of encoding said proteins, produce the DNA of the described variant of coding thus, express described DNA thereafter in the reconstitution cell culture.It is known being used for the technology that predetermined site at the DNA with known array carries out replacement mutation, for example mutation of M13 primer and PCR mutation.The normally single residue of amino acid replacement, but can take place at many diverse locations simultaneously; Insertion is typically about 1-10 amino acid residue; The scope of disappearance is about 1-30 residue.Preferably, lack or insert, promptly lack 2 residues or insert 2 residues contiguous residue centering.Displacement, disappearance, insertion or their any combination all can combine to obtain final construct.Described sudden change necessarily can not place sequence outside the reading frame, and preferably should not form the complementation district that can produce secondary mRNA structure.The displacement variant be those wherein at least one residue be removed and on its position, inserted the variant of different residues.That the displacement of this class is normally carried out according to hereinafter invading the exterior 3 and table 4 and be commonly called preservative replacement.

Table 3: amino acid abbreviations

Amino acid abbreviations

Alanine Ala A

Alloisoleucine Alle

(allosoleucine)

Arginine Arg R

Agedoite Asn N

Aspartic acid Asp D

Cysteine Cys C

Glutamic acid Glu E

Glutamine Gln Q

Glycine Gly G

Histidine His H

Isoleucine Ile I

Leucine Leu L

Lysine Lys K

Phenylalanine Phe F

Proline Pro P

Pyroglutamic acid pGlu

Serine Ser S

Threonine Thr T

Tyrosine Tyr Y

Tryptophan Trp W

Valine Val V

Table 4: amino acid replacement

The exemplary conservative substitution of former residue, other are as known in the art.

Ala ser

Arg lys，gln，his

Asn gln；his

Asp glu

Cys ser

Gln asn，lys

Glu asp

Gly Ala

His asn；gln

Ile leu；val

Leu ile；val

Lys arg；gln；his

Met Leu；ile

Phe met；leu；tyr

Ser thr，asn

Thr ser，gln

Trp tyr

Tyr trp；phe

Val ile；leu

Make function or immune system characteristic generation substantial variations by the displacement of selecting not compare conservatively with displacement shown in the table 3, it is the visibly different residue of selection, described effect is meant: (a) keep the polypeptide backbone structure in the replacement areas, for example as folding or helical conformation; (b) the electrical or hydrophobicity at maintenance molecule target site place or (c) the huge side chain of maintenance.It will be such displacement that common expection can make property of protein that the maximum displacement that changes takes place, and (a) hydrophilic residue in the described displacement (for example seryl-or Threonyl) is replaced by hydrophobic residue (for example leucyl-, isoleucyl-, phenylalanyl, valyl or alanyl); (b) cysteine or proline are replaced by other arbitrary residues; (c) residue (for example lysyl-, arginyl-or histidyl-) with electropositive side chain is replaced (for example glutamyl or aspartyl) by the elecrtonegativity residue; (d) residue (for example phenylalanine) with bulky side chain replace by the residue of unprotected side chain (in this case, for example glycine); (e) quantity of increase sulphation and/or glycosylation site.

For example, using another biology and/or chemically similar amino acid residue to replace an amino acid residue is exactly preservative replacement well known by persons skilled in the art.For example, preservative replacement can be to use another hydrophobic residue to replace a hydrophobic residue, or uses another polar residues to replace a polar residues.Described displacement comprises combination, for example Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; With Phe, Tyr.The version of the preservative replacement that this class is carried out every exemplary disclosed sequence is comprised in the chimeric polyeptides (mosaic polypeptide) that this paper provides.

Can adopt the displacement or deletion mutagenesis inserts or inactivation N-glycosylation site (Asn-X-Thr/Ser) or O-glycosylation site (Ser or Thr).The disappearance of cysteine or methionine or other unstable residues or displacement (for example in " neutrophil cell resistance " albumen because neutrophil cell produces oxidant) also may be required.The disappearance in potential Proteolytic enzyme site (for example Arg) or displacement can be by lacking an alkaline residue or realizing with glutaminyl or alkaline residue of histidyl-residue displacement.

It is described by the result of polypeptide expressed that some translation back derivatization is that recombinant host cell acts on.Glutaminyl and asparaginyl-residue are often become corresponding glutamyl and aspartyl by deamidate after translation.Perhaps, above-mentioned residue can be by deamidate under the acid condition of gentleness.Other post translational modifications comprise the (T.E.Creighton that methylates of amido in the epsilon-amino group of phosphorylation, lysine, arginine and histidine side chain of oh group of hydroxylating, seryl-or Threonyl residue of proline and lysine, Proteins:Structure and MolecularProperties, W.H.Freeman ﹠amp; Co., San Francisco pp 79-86[1983]), the acetylation of N-terminal amido, and the amidatioon of C-terminal carboxyl in some cases.

Disulfide bond is the covalent interaction between the mercapto groups of two cysteine molecules.By oxidation reaction, hydrogen atom is removed from mercapto groups, make to form disulphide bridges; The bonding cysteine of gained is called as cystine.Disulfide bond can classify I class and II class.The II generic key is by connecting the three dimensional structure that intrachain cysteine comes stabilize proteins.When above-mentioned interaction has just produced I class disulfide bond when different interchains takes place.The formation of I class disulfide bond can help to form dimer protein, this necessary a kind of key character when normally receptor provides suitable receptor-ligand binding.Amino acid replacement can carry out in the site that has cysteine residues; Usually preservative replacement can not change cysteine residues related in the disulfide bond.If participated in disulfide group by metathetical residue, then this class displacement may have the protein folding of change or change the interactional effect of polymer.Can determine which cysteine has participated in disulfide bond.

It should be understood that and can combine in the combination in any mode the description of conservative sudden change and homology for example have the example of at least 70% homology with particular sequence, wherein said variant is that conservative is suddenlyd change.

Discussed in this description various albumen and protein sequence it should be understood that the nucleic acid that also discloses these protein sequences of encoding.This should comprise the degenerate sequence that all are relevant with the specific protein sequence, and promptly all contain the nucleic acid of the sequence of a kind of specific protein sequence of encoding, and the variant of all codings this protein sequence disclosed herein and the nucleic acid (comprising degeneracy nucleic acid) of derivant.Therefore, though may not write out each concrete nucleotide sequence herein, it should be understood that this description by disclosed protein sequence in fact disclosure and description every kind and each sequence.Though it should also be understood that it is which kind of concrete DNA sequence this albumen of encoding that aminoacid sequence can not show in organism, but a kind of when disclosing proteic specific variants when disclosing herein, produce described that this proteic known nucleic acid sequence sequence of coding also is known and has carried out disclosure and description in this article in this proteic particular organisms.

The invention also discloses the fragment of disclosed albumen and variant.Common above-mentioned fragment will keep at least a function as herein described, for example enhanced blood-brain barrier permeability.For example it should be understood, however, that, still can use not keep this active fragment to generate antibody.For example should also be understood that, exist various by different functional activity that galanin kept.Above-mentioned activity can be correlated with but optional.The technical staff will appreciate that how by for example transforming the functional structure territory that disclosed analog is handled in the zone that can influence specific function.The analog that has removed or transformed the particular functionality site is disclosed among the embodiment 1 and 2.

8. antibody

Antibody disclosed herein can be used to differentiate the analog with required function.Term used herein " antibody " includes but not limited to the panimmunity globulin (being complete antibody) of any class.Natural antibody is allos tetramer glycoprotein normally, has two identical light (L) chains to form with two identical weights (H) chain.Usually, every light chain all links to each other with a heavy chain by a covalent disulfide bonds, and the quantity of described disulfide bond is variable between the heavy chain of different immunoglobulin isotypes.Every heavy chain and light chain also have equally distributed intrachain disulfide bond.Every heavy chain all at one end has a variable region (V (H)), is connected to many constant regions thereafter.Every light chain all at one end has a variable region (V (L)) and has a constant region at its other end; Described constant region of light chain and described heavy chain first constant region side by side, and described variable region of light chain and described variable region of heavy chain are side by side.It is believed that specified amino acid residues has formed the interface between described light chain and variable region of heavy chain.According to the aminoacid sequence of its constant region, all can be divided into two kinds of visibly different types from the light chain of any vertebrate antibody, be called kappa (k) and lamda (1).According to the aminoacid sequence of its CH, immunoglobulin can be divided into different types.Have five big class human normal immunoglobulin: IgA, IgD, IgE, IgG and IgM, and wherein a few class also can be divided into subclass (isotype), for example IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1 and IgA-2.Those skilled in the art can discern the corresponding classification in the mice.The CH corresponding with the inhomogeneity immunoglobulin is called as α, δ, ε, γ and μ respectively.

This paper uses term " variable " to describe some part of the variable region that sequence is different between the antibody, and determined combination and the specificity of every kind of specific antibodies at its specific antigen.Yet variability distributes usually and anisotropically in the whole variable region of antibody.In light chain and variable region of heavy chain, variability concentrates in three sections that are called as complementary determining region (CDR) or hypervariable region usually.The part of conservative Du Genggao is called as skeleton (FR) in the variable region.The variable region of natural heavy chain and light chain includes four FR districts, mainly takes the βZhe Die configuration, is connected by three CDR, and this can form the ring (forming the part of described βZhe Die structure in some cases) that connects described βZhe Die structure.CDR in every chain is all closely combined by described FR district, and form the antigen binding site of antibody (referring to Kabat E.A.et al. with the CDR of other chain, ＂ Sequences of Proteins of ImmunologicalInterest, ＂ National Institutes of Health, Bethesda, Md. (1987)).Constant region is not participated in antibody directly and is combined with antigenic, but shows various effector functions, for example participation of antibody in the antibody dependent cellular cytotoxicity.

Term described herein " antibody " or its fragment comprise chimeric antibody and hybrid antibody and the fragment with two or more antigens or epitope specificity, and for example F (ab ') 2, Fab ' and Fab etc. comprise hybridized fragment.Therefore, provide the antibody fragment that has kept with the bonded ability of its specific antigen.For example, kept the antibody fragment of enhanced permeability all to belong in the intended scope of term " antibody or its fragment ".This antibody-like and fragment can utilize technology as known in the art to prepare, and can screen (referring to Harlow andLane.Ant ibodies it according to the conventional method that is used to produce antibody and screen antibody with regard to specificity and activity, A Laboratory Manual.Cold Spring HarborPublications, New York, (1988)).

Comprise that also for example United States Patent (USP) 4,704 in the implication of " antibody or its fragment ", the conjugate of antibody fragment described in 692 and antigen-binding proteins (single-chain antibody), the content of described list of references mode is by reference included this description in.

9. pharmaceutical carrier/drug products sends

As indicated above, described compositions (for example galanin analog) can also be come vivo medicine-feeding with pharmaceutically suitable carrier." pharmaceutically acceptable " is meant and is not biologically or the undesirable material in other aspects, be that described material can give the experimenter with nucleic acid or carrier, and can not cause any undesirable biotic influence or with harmful mode and any other component interaction that contains the pharmaceutical composition of this material.Certainly should screen so that any degraded of described active component is all minimum and make the intravital any harmful side effect minimum of described experimenter described carrier, be known in those skilled in the art like this.

Described compositions can be for oral administration, parenteral (for example intravenous), by the intramuscular injection administration, by the peritoneal injection administration, through skin administration, treated in vitro, topical etc., and can use local intranasal administration or by the inhalant administration.The accurate amount of desired composition will change to some extent according to experimenter's difference, depend on experimenter's species, age, body weight and ordinary circumstance, used specific nucleic acid or carrier, its mode of administration etc.Therefore, can not stipulate the accurate amount of each compositions.Yet the normal experiment method that those skilled in the art only use instruction herein to provide can be determined the amount that is fit to.

If necessary, the parenteral of described compositions is normally undertaken by injection.Injection conventionally form be can be made, liquid solution or suspensions, solid form or emulsion form that liquid forms suspension are suitable for before injection, being dissolved in.A kind of method of revising recently that is used for parenteral comprises uses slow release or sustained release system so that can keep constant dosage.Referring to, for example United States Patent (USP) 3,610,795, and described list of references mode is by reference included this description in.

Described material can form suspension (for example including in microparticle, liposome or the cell) in solution.Above-mentioned substance can come the specific cell type of targeting by antibody, receptor or receptors ligand.Following list of references is to use this technology to make the example of specific protein target tumor tissue

(Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagsh awe, K.D., Br.J.Cancer, 60:275-281, (1989); Bagshawe, et al., Br.J.Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog.Reviews, 129:57-80, (1992); And Roffler, et al., Biochem.Pharmacol, 42:2062-2065, (1991)).

Carrier for example the liposome (lipid that comprises mediation drug targeting colon carcinoma) puted together of " stealth " and other antibody, the receptor by cell specific ligand mediated dna targeting, instruct the lymphocyte of cancer target, and the treatment retrovirus retrovirus of the high degree of specificity of targeted mouse glioma cell in vivo.Following list of references be to use this technology make specific protein target tumor tissue example (Hughes et al., Cancer Research, 49:6214-6220, (1989); With Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)).Generally speaking, the endocytosis approach can relate to receptor (composing type or part induction type).Above-mentioned receptor clustering enters cell by clathrin-coated vesicle in clathrin-coated pit, pass describedly to be known from experience therein by the acid endosome of sorting, is re-circulated to cell surface then and is stored in the born of the same parents or is degraded in lysosome.The internalization approach can be brought into play various functions, for example nutrition intake, the conditionality of the removing of activated protein, macromolecular removing, virus and toxin enter, the dissociating and degrading of part, and the adjusting of acceptor levels.Many receptors are followed approach in a plurality of born of the same parents, depend on cell type, acceptor density, part type, part atomicity and ligand concentration.The molecule of receptor-mediated endocytosis and cell mechanism in following list of references, obtained the summary (Brown and Greene, DNA and Cell Biology10:6,399-409 (1991)).

A) pharmaceutically suitable carrier

Described compositions (comprising the galanin analog) can be used for the treatment of with pharmaceutically suitable carrier.

Pharmaceutical carrier is well known by persons skilled in the art.Above-mentioned carrier great majority normally are used for the standard vector with the medicine administration of human, comprise for example buffer solution of sterilized water, saline and physiological pH of solution.Described compositions can be intramuscular administration or subcutaneous administration.Other chemical compounds will come administration according to the standard method that those skilled in the art use.

Except that selected molecule, pharmaceutical composition also may comprise carrier, thickening agent, diluent, buffer, antiseptic, surfactant etc.Pharmaceutical composition also can comprise one or more active component, for example antimicrobial, antiinflammatory, anesthetis etc.

Described pharmaceutical composition can be with multiple mode administration, depends on that needing topical therapeutic still is whole body therapeutic and the zone that will treat.Administering mode can be topical (comprising administration through eye, vagina administration, rectally, intranasal administration), oral administration, pass through inhalation or parenteral, for example by intravenous drip administration, subcutaneous administration, intraperitoneal administration or intramuscular injection.Disclosed compositions (for example galanin analog) can be for intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, intracavitary administration or through the skin administration.

The preparation that is used for parenteral comprises sterile aqueous or non-aqueous solution, suspension or emulsion.The example of non-aqueous solvent is a for example olive oil of propylene glycol, Polyethylene Glycol, vegetable oil, and injectable organic ester ethyl oleate for example.Aqueous carrier comprises water, alcohol/aqueous solution, emulsion or suspension, contains salt or buffer medium.The parenteral carrier comprises sodium chloride solution, woods Ge Shi glucose, glucose and sodium chloride, Ru Suanlingeshi oil or fixed oil.Intravenous vehicles comprises liquid and supplementary, electrolyte replenisher (for example those are based on the supplement of woods Ge Shi glucose) etc.Can also there be antiseptic and other additives, for example antibacterial, antioxidant, chelating agen and noble gas etc.

The preparation that is used for topical can comprise ointment, lotion, emulsifiable paste, gel, drop, suppository, spray, liquid and powder.Conventional pharmaceutical carrier, aqueous base, powder dress alkali or oily alkali, thickening agent etc. are essential or desirable.

Be used for liquid preparations for oral administration and comprise powder or granule, suspension or aqueous solution or non-aqueous media, capsule, wafer or tablet.Thickening agent, flavoring agent, diluent, emulsifying agent, dispersing aid or binding agent are desirable.

Some compositions may be with the form administration of pharmaceutically acceptable acid addition salts or base addition salts, described salt is by forming with mineral acid or organic acid or inorganic base or organic base reaction, described mineral acid is hydrochloric acid for example, hydrobromic acid, perchloric acid, nitric acid, Hydrogen thiocyanate, sulphuric acid and phosphoric acid, described organic acid is formic acid for example, acetic acid, propanoic acid, glycolic, lactic acid, acetone acid, ethanedioic acid, malonic acid, succinic acid, maleic acid and fumaric acid, described inorganic base is sodium hydroxide for example, ammonium hydroxide, potassium hydroxide, described organic base is monoalkylamine for example, dialkylamine and trialkylamine, aromatic amine and through metathetical ethanolamine.

B) therapeutic use

The dosage scope of described compositions is that the symptom of wherein said obstacle is affected even as big as producing required effect.Described dosage should be greatly to causing deleterious side effect, for example unwanted cross reaction, anaphylactic reaction etc.Generally speaking, described dosage can change along with patient's age, situation, sex and disease degree, and can be determined by those skilled in the art.In any contraindication incident, every doctor all can adjust described dosage.Dosage is variable, and can give one or many dosage every day, continues one to a few days.

10. chip and microarray

The invention discloses at least one address wherein and be the chip of the part of sequence listed in arbitrary nucleotide sequence disclosed in this invention or sequence.The invention also discloses at least one address wherein and be the chip of the part of sequence listed in arbitrary peptide sequence disclosed in this invention or sequence.

The invention also discloses at least one address wherein and be the chip of variant of the part of sequence listed in arbitrary nucleotide sequence disclosed in this invention or sequence.The invention also discloses at least one address wherein and be the chip of variant of the part of sequence listed in arbitrary peptide sequence disclosed in this invention or sequence.

The invention also discloses at least one address wherein and be the chip of the part of sequence listed in arbitrary nucleotide sequence disclosed in this invention or sequence, wherein said sequence comprises at least one variant sequence disclosed herein.The invention also discloses at least one address wherein and be the chip of the part of sequence listed in arbitrary peptide sequence disclosed in this invention or sequence, wherein said peptide sequence comprises at least one galanin analog disclosed herein.

The invention also discloses at least one address wherein and be the chip of the part of sequence listed in arbitrary nucleotide sequence disclosed in this invention or sequence, wherein said sequence comprises at least one variant sequence in the zone of this paper definition.The invention also discloses at least one address wherein and be the chip of the part of sequence listed in arbitrary peptide sequence disclosed in this invention or sequence, wherein said peptide sequence comprises at least one displacement disclosed herein, addition, sudden change or disappearance.

11. computer-readable medium

It should be understood that disclosed nucleic acid and albumen can be represented as the sequence of being made up of nucleotide or aminoacid.The mode that has the above-mentioned sequence of various demonstrations, for example the nucleotide guanosine can be represented as G or g.Similarly, the aminoacid valine can be represented as Val or V.It will be understood by those skilled in the art that as any in the various known ways as described in how and show and express any nucleic acid or protein sequence that every kind of mode all should be considered to disclosed herein.This paper has considered especially and shown above-mentioned sequence, for example commercially available floppy disk, tape, chip, hard disk, CD and optic disc, or other computer-readable mediums on computer-readable medium.The invention also discloses described open sequence with binary code representation.It will be understood by those skilled in the art that what is a computer-readable medium.Therefore, the computer-readable medium that has wherein write down, preserved or preserved described nucleic acid or protein sequence is disclosed.

The invention discloses the computer-readable medium that contains listed sequence of this paper and sequence relevant information.

12. test kit

The invention discloses the test kit that contains the reagent that can be used to implement method disclosed herein.Described test kit can comprise any reagent as herein described or agent combination or should be understood that is any reagent or the agent combination that need be used for or help to implement method disclosed herein.For example, described test kit can comprise the metathetical aminoacid described in some embodiment that is used to carry out described method, and operation instructions.

13. have the compositions of similar functions

It should be understood that compositions disclosed herein has some function, for example enhanced blood-brain barrier permeability.Herein disclosed is some structural requirements that are used to realize disclosed function, and it should be understood that and have multiple realize identical function and structure disclosed structurally associated, and said structure obtains identical result the most at last.

E. prepare described method for compositions

Unless otherwise specified, compositions disclosed herein and implement the required compositions of disclosed method and can use any method that is used for this particular agent or chemical compound well known by persons skilled in the art to prepare.It should be understood that, except as otherwise noted, conventional Protocols in Molecular Biology can be used for preparing disclosed molecule and implements described disclosed method, described Protocols in Molecular Biology is Sambrooket al. for example, MolecularCloning:A Laboratory Manual, 2nd Edition (ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., disclosed those technology 1989).

The present invention discloses the method for compositions that a kind of preparation has enhanced blood-brain barrier permeability especially, comprise that preparation has a compositions of enhanced blood-brain barrier permeability, wherein said compositions comprises a kind ofly compares the peptide with enhanced lipophilic characteristic and enhanced alkalescence with this peptide of not transformed.In an example, described lipophilic characteristic can be strengthened by described peptide and hydrophobic part (for example poly-aliphatic chain) are puted together.Described lipophilic characteristic can also be strengthened by the halogenation that increases aromatic moieties.Described alkalescence can be by introducing positively charged the same oligomer and the different oligomer of amino acid residue strengthened, the amino acid residue of described positively charged includes but not limited to the ornithine of lysine, arginine, high-lysine, homoarginine, L-or D-isomer configuration; 2, the 3-diaminopropionic acid; 2,4-diamino-butanoic.In another example, described alkalescence can be by being strengthened with puting together based on the part of polyamines, and described part based on polyamines is spermine, spermidine, polyamide-amide type dendrimer or polyamines toxin and derivant thereof for example.Described peptide with do not compared by this peptide of forms of modification, the efficient of passing blood brain barrier can improve 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.When not compared by this peptide of forms of modification, described peptide can also have the glycosylation of increase.Described peptide can contain sept.Described sept is selected from: Gly, Ahx, Gly-Ahx or PEG-O20c.

1. nucleic acid is synthetic

For example, described nucleic acid (oligonucleotide that for example is used as primer) can use the chemical synthesis process of standard to prepare, and perhaps can use Enzymology method or any other known method to produce.The scope of these class methods contains from the enzymic digestion of standard and adds that nucleotide fragments separates (referring to for example, Sambrook etal., MolecularCloning:ALaboratoryManual, 2nd Edition (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) Chapters 5,6) to pure synthetic method, (for example for example use Milligen or Beckman System 1Plus synthesizer, Model 8700 automated synthesizer of Milligen-Biosearch, Burlington, MA or ABI Model 380B) the cyanoethyl phosphoramidite method.Ikuta et al., Ann.Rev.Biochem.53:323-356 (1984) (phosphotriester and tris phosphite method) and Narang etal., Methods Enzymol, 65:610-620 (1980) (phosphotriester method) has also described the synthetic method that can be used for preparing oligonucleotide.(peptide nucleic acid(PNA) molecule) can use known method to prepare, Nielsen et al. for example, the method described in the Bioconjug.Chem.5:3-7 (1994).

2. peptide is synthetic

It is a kind of that to be used to produce disclosed proteic method be to utilize the protein chemistry technology that two or more peptides or polypeptide chain are connected together.For example, peptide or polypeptide can be to use present available laboratory equlpment utilize Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tertbutyloxycarbonyl) chemical agent (Applied Biosystems, Inc., Fos ter City, CA) chemically synthetic.Those skilled in the art can easily understand with corresponding peptide of disclosed albumen or polypeptide and can synthesize by the chemical reaction of standard.For example, can synthesize peptide or polypeptide and it is not cut down from its synthetic resin, and can synthesize peptide or proteic other fragments and subsequently it is cut down from resin, thereby expose function by the end group of described other fragments blocking-up.By the peptide condensation reaction, at above-mentioned two segmental carboxyls and amino terminal they are covalently coupled together respectively via peptide bond and to form albumen or its fragment (Grant GA (1992) Synthetic Peptides:A User Guide.W.H.Freeman and Co., N.Y. (1992); Bodansky M and Trost B., Ed. (1993) Principles of PeptideSynthesis.Springer-Verlag Inc., NY (relate to the synthetic data of peptide mode by reference in the described list of references at least and include this description in).Perhaps, can be according to synthetic in vivo separately described peptide described herein or polypeptide.In case separate, can by similar peptide condensation reaction will above-mentioned independent peptide or polypeptide couple together formation peptide or its fragment.

For example, the enzyme of clone or section of synthesized peptide connects feasible generation bigger fragments of peptides, polypeptide or the whole protein structure domain (Abrahmsen L et al., Biochemistry, 30:4151 (1991)) of short relatively fragments of peptides can being coupled together.Perhaps, can utilize the short big peptide or the polypeptide of the synthetic property of fragments of peptides ground structure of native chemical connection cause of synthetic peptide.This method constitutes (Dawson et al.Synthesis of Proteins by Native Chemical Ligation.Science, 266:776-779 (1994)) by two step chemical reactions.The first step be a unprotected synthetic peptide-thioesters with another contain carry out between the unprotected peptide section of amino terminal Cys residue the chemo-selective reaction with the intermediate product that produces the mercapto ester and be connected as initial covalency product.Under the situation that does not change reaction condition, described intermediate product can experience and react between spontaneous rapid molecular so that form natural peptide bond (Baggiolini M et al. (1992) FEBS Lett.307:97-101 in connection site; Clark-Lewis Iet al., J.Biol.Chem., 269:16075 (1994); Clark-LewisIetal., Biochemistry, 30:3128 (1991); Rajarathnam K et al., Biochemistry33:6623-30 (1994)).

Perhaps, unprotected peptide section can be connected by chemical ground, and this moment, the formation key was non-natural (non-peptide) key (Schnolzer, M et al.Science, 256:221 (1992)) between the described peptide section because chemistry is connected.This technology has been used for synthetic proteins domain analog and has purifiedly relatively in a large number had a complete bioactive albumen (deLisle Milton RC et al., Techniques in ProteinChemistry IV.Academic Press, New York, pp.257-267 (1992)).

3. be used to prepare described method for compositions

The invention discloses and be used to prepare the method that described compositions and preparation can produce the intermediate product of described compositions.For example, the invention discloses albumen among the SEQ ID NO:1-55.There is the multiple method that can be used to prepare above-mentioned composition, for example synthetic chemistry method and standard molecular biology method.It should be understood that the above-mentioned and other disclosed compositions method of preparation is by open particularly.

The invention discloses the albumen of producing by following method, described method comprises: the nucleic acid of a coding galanin analog is operably connected with the sequence of a described expression of nucleic acid of regulation and control, and described galanin analog comprises the sequence of listing among the SEQ ID NO:3.

The invention also discloses the albumen of producing by following method, described method comprises: the nucleic acid molecules of a coding galanin analog is regulated and control described nucleic acid and is operably connected with one, described galanin analog comprise with SEQ ID NO:3 in the sequence listed have the sequence of 80% homogeneity.

The invention discloses the cell of producing by the method for using any disclosed nucleic acid transformant.The invention discloses the cell of producing by the method for using the disclosed nucleic acid transformant that any non-natural exists.

The invention discloses utilization and express any disclosed peptide that the method for any disclosed nucleic acid is produced.The invention discloses the disclosed peptide that any non-natural that method that utilize to express any disclosed nucleic acid produces exists.The invention discloses utilization and express any disclosed peptide that the method for any non-natural disclosed nucleic acid is produced.

The invention discloses the animal that produces by the method for using any nucleic acid molecules transfection animal body inner cell disclosed herein.The invention discloses the animal that produces by the method for using any nucleic acid molecules transfection animal body inner cell disclosed herein, wherein said animal is a mammal.The invention also discloses the animal that produces by the method for using any nucleic acid molecules transfection animal body inner cell disclosed herein, wherein said mammal is mice, rat, rabbit, cattle, sheep, pig or primates.

The invention also discloses utilization any cell disclosed herein is joined the animal that the intravital method of described animal is produced.

It should be understood that and another kind of produce described proteic method and can use the rabbit expression system, for example the system of those types of producing by Bioprotein Technologies.Disclosed molecule can use the carrier of the above-mentioned type and production system to produce.For example, the system of the above-mentioned type is for disclosed (EPO patent application N ° 92 401 635.5, United States Patent (USP) 5,965,788) and be positioned on the gene separaant (EPO patent application N ° 00 403 658.8), and information can be in network address Www.bioprotein.comFind.

The application in the whole text in, quoted various publications.The whole modes by reference of the disclosure of these publications are included the application in, so that more fully describe the situation in the affiliated field of the present invention.The data that is comprised in the disclosed list of references also individually and particularly by reference mode include this description in, described data is discussed in the critical sentence of described list of references to some extent.

It will be apparent to those skilled in the art that can modifications and variations of the present invention are and do not deviate from scope of the present invention or spirit.Consider description of the present invention disclosed herein and enforcement, other embodiments of the present invention will be conspicuous for the art technology people.It is exemplary that described description and embodiment only should be considered to, and following claim illustrates true scope of the present invention and spirit.

F. embodiment

Provide the following example so that for those of ordinary skills provide about how preparing and assess the complete disclosure and description of described herein and claimed compounds, compositions, goods, equipment and/or method, and be intended to illustrate the present invention purely and be not that the scope of the present invention that is intended to the inventor is thought limits.Endeavoured to ensure the accuracy of numeral (for example quantity, temperature etc.), can some mistakes of existence and deviation but should consider.Except as otherwise noted, part is meant weight portion, temperature for ℃ unit or refer to room temperature, and pressure is meant atmospheric pressure or near atmospheric pressure.

1. embodiment 1: have systemically active anticonvulsant galanin analog

For obtaining to be transformed to strengthen the Proof of Concept result of its blood brain barrier penetrance, two kinds of neuropeptides have been selected: somatostatin and galanin about the anticonvulsant neuropeptide.As indicated above, above-mentioned two kinds of neuropeptides all have anti-convulsant activity.

Overall experimental technique is shown in Figure 7.Designed and synthesized one group of neuropeptide analog (the 1st generation) to test them with the ability of high-affinity in conjunction with its each autoreceptor.The described group of about 10 kinds of analog that comprise every kind of neuropeptide.Further test high-affinity analog penetrates the ability of blood brain barrier.Synthetic and assessment second filial generation analog is third generation analog then after the result who obtains first generation analog.Select most promising analog (high-affinity part) to confirm their agonist activity by functional examination with enhanced blood-brain barrier permeability.In vivo a subclass (the effective agonist with enhanced blood-brain barrier permeability) of above-mentioned analog is carried out pharmacology's test then.

For as medicine, the neuropeptide analog should have several important properties, comprising: (1) height is tired and selectivity, (2) metabolic stability, (3) relative long half-life and the body loop cleaning rate of reduction, and (4) enhanced blood-brain barrier permeability.Most of neuropeptides demonstrate high tiring and selectivity.Metabolic stability can not replaced responsive residue by the residue that proteolytic enzyme is discerned by peptide backbone modifications and/or use usually and be introduced.Can realize the increase of half-life and the reduction of eliminating speed effectively by a part based on polymer is puted together (for example, Pegylation) with peptide.Higher blood-brain barrier permeability can pass through to increase lipotropy or cationization, and introduces by adding prodrug, nutrition transhipment analogies or glycosylation.The structure of ideal medicine neuropeptide illustrates in Fig. 8 schematically.

As shown in Figure 8, new ideas in the neuropeptide engineering have been introduced: " BBB/PK is modified ".Described BBB/PK is modified comprise have lipophilic, cation and the unitary huge part of transhipment analogies based on polymer; This is modified to have dual purpose, strengthens blood-brain barrier permeability and improves pharmacokinetic property.Described cation and lipophilic unit can promote respectively and negative electricity film surface between interaction and raising stride film diffusion.The function of described active transport model configuration is to improve the specificity of brain capture neuropeptide by enhancing and the interaction between the specificity nutrition transport protein on the brain surface epithelial cell.The structural framing that contains whole said units can also improve the pharmacokinetic property of described peptide, the effect of the peg moiety that simulation/replacement is commonly used.Test is as the N of described model neuropeptide or the above-mentioned huge part of C-terminal extension, and this paper also discloses more general link position in the described neuropeptide structure.

Use following method design to have the neuropeptide analog of enhanced blood-brain barrier permeability: if possible, to use the analog of metabolic stability to begin.Differentiate and to carry out metathetical other AA positions of side chain in the described analog.Discriminating is positioned at the position that N and C-terminal can be introduced huge part.Replace lipotropy and the alkalescence that increases analog by side chain.Introduce extension to peptide analogues, described extension will further increase its lipotropy and alkalescence, improves pharmacokinetic property (BBB/PK is modified) simultaneously.Comprise that at described extended spot a nutrition model configuration is to improve the specificity that blood brain barrier penetrates.Make up described analog and described extension (BBB/PK is modified) that side chain is modified that have.

The key that success designs this class analog is the correct combination of above-mentioned modification.For realizing this goal, when each set that designs and assess modification and their best of breed, can adopt system approach.Group method illustrates in Figure 10 schematically.Disclosed hereinly can in the solid-phase peptide building-up process, use the automatic peptide synthesizer to introduce to amino acid whose modification.All alpha-non-natural amino acids or put together the derivant that structure all is commercially available Fmoc protections.

Somatostatin is a kind of 14 amino acid whose hypothalamus peptides that contain a single disulfide bond, is found in 1973 at first (Brazeau et al., 1973).The sequence of somatostatin is as described below:

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Ala Gly Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys

SAR research has widely had been found that 5 Key residues: Phe ⁶, Phe ⁷, Trp ⁸, Lys ⁹And Phe ¹¹, and use alanine displacement Gly ², Lys ⁴, Asn ⁵, Thr ¹⁰, Thr ¹²Or Ser ¹³Can obviously not influence biological activity (Vale et al., 1975).In addition, proved and contained D-Trp ⁸Analog more effective because its Proteolytic enzyme resistance is stronger and/or activity conformation is more stable.

[D-Trp ⁸] somatostatin can be used as the analog of the metabolic stability that is used for method disclosed herein.In order to increase alkalescence, but Thr, Ser or Asn residue can be by similar positively charged DAB of isosterism (DAB) or the systematically displacements of DAP (diaminopropionic acid) residue institute.For increasing lipotropy, can introduce the Lys-palmityl and partly replace Lys ⁴Or Asn ⁵, and/or use the halogenation equivalent chloro-Phe residue of Phe residue to replace the Phe residue.As summing up in the table 5, synthesized nine kinds of analog and measured them and the affinity of the somatostatin receptor.With combination does not cause the modification of negative influence to combine to high-affinity.Above-mentioned second filial generation analog contains the modification of 2-4 kind combination.

Next, the N-terminal extension is incorporated into [D-Trp ⁸] in the somatostatin.Above-mentioned extension (as shown in Figure 8, BBB/PK is modified) have two purposes: (1) utilizes passive and active mechanisms to improve blood-brain barrier permeability and (2) to improve the pharmacokinetic property of neuropeptide medicine by adding the huge part (bulky moiety) that can reduce clearance rate and improve the proteolytic degradation resistance.Because this class " BBB/PK is modified " is a new notion, therefore use several compositionss of the construction unit of rare several formation extensions.Table 6 provides the unitary 26S Proteasome Structure and Function information about being recommended.

Table 5The metathetical summary of side chain in the somatostatin that proposes in this research

DAB: DAB; DAP: diaminopropionic acid; The Lys-palm:Lys-palmityl; Cl-Phe:Chloro-phe.

Table 6 is used for the summary of the modified unitary 26S Proteasome Structure and Function character of synthetic BBB/PK

The unit	Structure	Function/note
			AHX	Aminocaproic acid	Increase the described lipotropy that extends the middle part and stride with enhancing that film is passive invades

		Go into.Do not introduce extra hydrogen bond donor/receptor.
			PerFHX	The perfluor caproic acid	By adding medicated cap to increase lipotropy at N-terminal with extremely hydrophobic " tail ".This is a kind ofly to increase hydrophobicity and the very effective method of the described extended molecule size of not obvious increase.
The PEG-sept	8-amino-3,6-two oxa-s are sad	Use PEG base sept to increase the length/size of described extension: this will improve the pharmacokinetic property of described analog.
			Phe	Phe-[D-Phe]	Phe analogies as the identification of nutrient active transport substrate; Perhaps, also may introduce glycosyl part.
Few-(Lys)	Lys-(D-Lys)-Lys-(D-Lys)-Lys-(D-Lys)-Lys	Increase the alkalescence of described extension: this should be able to strengthen the electrostatic interaction with film.

Described unit can be introduced in the solid phase synthesis process with as to [D-Trp ⁸] Ala of somatostatin ¹The extension of residue.Table 7 and table 11 have been summed up described extension.

The summary (employed abbreviation is with reference to table 6) that N-terminal extends in table 7 somatostatin analogs

Analog #	Unit	3	Unit 2	Unit 1	Analog
						EXT1			AHX-AHX	[D-Trp 8]SOM
EXT2			PerFHX	[D-Trp 8]SOM
					EXT3			The PEG-sept	[D-Trp 8]SOM
EXT4		AHX-AHX	The PEG-sept	[D-Trp 8]SOM
					EXT5		PerFHX	The PEG-sept	[D-Trp 8]SOM
EXT6		Oligo-(Lys)	The PEG-sept	[D-Trp 8]SOM
					EXT7		Phe	AHX-AHX	[D-Trp 8]SOM
EXT8		Phe	AHX	[D-Trp 8]SOM
					EXT9	AHX-AHX	Oligo-(Lys)	The PEG-sept	[D-Trp 8]SOM
EXT10	Phe	Oligo-(Lys)	The PEG-sept	[D-Trp 8]SOM

Synthesized 10 kinds of analog at first, and verified with regard to the associativity of itself and described somatropin receptor that it is assessed.Further assess the penetrating character of high-affinity analog by described model blood-brain barrier permeability algoscopy.

In case selected best extension, they just can be connected to has contained on the metathetical somatostatin analogs of the side chain of optimizing (referring to table 5).Owing to be difficult to the best of breed of prediction " extension analog " and " side chain displacement analog ",, wherein use every kind of selected extension to synthesize every kind of selected analog so adopt matrix method.During taking turns, this can obtain 9-12 kind analog.This class the 3rd generation analog can be tested with following whole three kinds of external tests: the permeability that (1) is attached on the described the somatostatin receptor, described blood brain barrier model is passed in (2) agonist activity and (3).Can select the most promising analog of limited quantity to be used for that the mice epilepsy model carries out pharmacology's test in described body.

Table 8 is used to design and has the matrix method that is connected with the modified somatostatin analogs of N-terminal BBB/PK.Wherein with three kinds of selected extended structures and three kinds of selected " side chain displacement " analog combinations.

	EXT-A	EXT-B	EXT-C
				Analog
1	√	√	√
				Analog 2	√	√	√
Analog 3	√	√	√

The similar method of the method that is used for somatostatin a kind of and mentioned above can be used to galanin and analog thereof.Galanin is a kind of 30 amino acid whose neuropeptides, but SAR studies show that with full-length peptide and compare that N-terminal partly remains very effective agonist (Langel and Bartfai, 1998).Galanin (1-16) analog can use jointly with method disclosed herein, and is wherein as follows, the Gly1 residue by N-methyl-Gly (sarcosine, SAR) replace:

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro His Ala Val

Gly ¹The methylate N-terminal that makes described peptide of N exempt from the proteolytic degradation of acceleration, and don't can obviously change its affinity (Rivera Baeza et al., 1994) to galanin receptors.SAR studies show that following residue is most important for biological activity: Gly ¹, Trp ², Asn ⁵, Tyr ⁹And Gly ¹²(Landet al., 1991).Samely discover that the N-terminal extension can cause bioactive forfeiture.On the other hand, when the C-terminal of galanin (1-16) partly is connected on the bigger structure, its as if can become highly stable (robust) (Pooga et al., 1998).Therefore, when only considered amino acid is replaced, [Sar ¹] method for designing of galanin analog is similar to the method that is used for somatostatin, but this method is introduced at C-terminal rather than N-terminal and is extended.Table 9 has been summed up the galanin analog that has amino acid replacement.

Table 9[Sar ¹] each residue replacement amino acid coding in the galanin (1-16) please refer to table 5.

C-terminal extends can be identical with the extension shown in the table 7, but at His ¹⁴Introduce the position.The synthetic then Lys that has ¹⁴(Mmt) (the Sar of residue (with the side chain of 4-methoxy trityl group protection) ¹) galanin.In coupling after the sarcosine, described peptide resin was handled 30 minutes with 1% TFA that is dissolved in dichloromethane.Lys ¹⁴The pendant amino group of residue can be by deprotection, then the described extension apparatus of coupling.Have displacement of bonded side chain and C-terminal extension the 3rd generation analog design identical with the described design that is used for somatostatin analogs.

The described peptide of the chemosynthesis of neuropeptide can use based on the solid-phase peptide synthetic method of Fmoc and automatic peptide synthesizer and synthesize.Carry out coupling method and described peptide is removed from solid support according to Chan and White (Chan and White, 2000) is described.By using reagent K to handle described peptide is removed from solid support.After through flushing and precipitation, described analog can use preparation type reversed-phase HPLC partition method to come purification.As Chen et al., 2000 is described, can form the disulfide bond in the described somatostatin analogs by the peptide of hatching purification with 2% DMSO, 30% acetic acid aqueous solution under the situation of pH7.0.Utilize this method can produce every kind of peptide analogues of several milligrams at least.

Blood-brain barrier permeability for assessment neuropeptide analog can adopt pION ' s PAMPA method.In the method, a filter that has fixed artificial membrane is placed between two donors and the receptor compartment.Described analog can place the donor compartment.After through the reasonable time interval, can use the UV spectrophotometer to come described donor and receptor compartment are carried out quantitatively.

Somatostatin and galanin are implemented by NovascreenBiosciences Corporation in conjunction with measuring (nonselective).In said determination, use rat forebrain film and radiolabeled parental generation neuropeptide.With single concentration (1 μ M) described analog is tested to distinguish the analog of high-affinity and low-affinity.The agonist activity of selected somatostatin and galanin analog can use the functional examination that is provided by MDS-Pharma Services further to test.(1 μ M) tests described analog with single concentration.

Convulsion test anti-convulsant activity can be determined in the Frings of AGS susceptible reflex epilepsy mouse model.AGS susceptible mice is the ideal acute epilepsy model that is used to carry out initial concept checking research because it be indifference and can effectively find various CNS reactive compounds (White etal., 1992).Assess have active peptide in Frings mice body with regard to its ability that prevents epilepsy, described epilepsy is that to utilize maximal electroshock (MES) and subcutaneous (s.c.) to give pentylenetetrazole (PTZ) inductive.Epilepsy chemical compound prevention epilepsy diffusion of being studied and the ability (White et al., 2002) that improves the epilepsy threshold value have been measured in above-mentioned two kinds of tests respectively.In case proved that with one or more of above-mentioned three kinds of epilepsies test a kind of modified peptide has activity, then the peak effect after the i.v. administration of formerly determining is carried out complete dose response study constantly.The effect research that carries out after result that above-mentioned Proof of Concept is studied and Intraventricular (i.c.v.) administration compares then.I.p. moving to left in the dose response curve can think to have realized that higher blood brain barrier penetrates.The result of above-mentioned three kinds of epilepsy test provides important data jointly, and described data have been supported the described method that makes the easier arrival brain of little peptide after the whole body administration.Hereinafter describe every kind of independent epilepsy test in detail.

Give the neuropeptide analogEach the modified neuroactive peptide that uses 10 μ l Hamilton syringes will be dissolved in the artificial cerebrospinal fluid of 5 μ l gives through Intraventricular (i.c.v.), and each modified neuroactive peptide that perhaps will be dissolved in 0.5% methylcellulose gives through intravenous (i.v.) according to every g body weight 0.01ml.

AGSAssess various modified peptides prevent AGS in the Frings of AGS susceptible mouse model when peak effect ability (White et al., 1992).For carrying out this test, each mice placed be equipped with audio sensor (Model AS-ZC; FETResearch and Development, Salt Lake City, (diameter 15cm in lucite tube UT); And be exposed under the sonic stimulation of 110 decibels (11KHz) continuing 20 seconds high 18cm).AGS is characterised in that bolts, and is the righting reflex forfeiture with forelimb and THE then.Think that the mice that does not show THE is to be subjected to protection.

The MES testFor carrying out the MES test, can before placing Corneal electrode, an anesthesia/electrolyte solution (being dissolved in 0.9% brinish 0.5% tetracaine hydrochloride) be added drop-wise in the eye of every animal.Electricity irritation in the mice MES test is for utilizing the 50mA that continues 0.2 second a kind of and equipment output like the initial described equipment class of Woodbury and Davenport (Woodbury andDavenport, 1952).With the disappearance of the tetanic property of the hind leg of epilepsy extensor component as terminal point.

Minimum toxotestUtilize swingle method (Dunham and Miya, 1957) in the mice body, to differentiate minimum toxicity.When mice being placed speed is on 1 inch groove rasp bar of 6r.p.m. the time, and this animal can be kept its balance for a long time.If described animal fell three times from this swingle, think that then it is poisoned in 1 minute.

Determine median effective dose (ED ₅₀ ) or half toxic dose (TD ₅₀ )Formerly the TPE of Que Dinging carries out convulsion/toxicity research in all quantitative bodies.Containing each groups of at least 8 mices with the test of the peptide of various dosage, is minimum toxic boundary between establishment at least two points until the protection that is minimum toxicity and 0% in 100% protection.Utilize drug dose (ED required when in 50% animal body, producing required end points in the every kind of test of computer program calculating based on the described method of Finney (Finney, 1971) then ₅₀Or TD ₅₀), 95% confidence interval, the slope of the regression line and the S.E.M. of slope.

The analog of somatostatin can see Table 10 (all analog all have the disulfide bond that forms between two cysteine residues):

Table 10:

SOM-BBB1	(NN3APG)(AHX)AGCKNFFWKTFTSC(SEQ ID NO：41)
		SOM-BBB2	(NN3APG)(AHX)AGCKNFF( DW)KT(Cl-Phe)T(Dap)C(SEQ ID NO：42)
SOM-BBB3	W(AHX)KKCKNFF( DW)KT(Cl-Phe)(Dab)(Dab)C(SEQ ID NO：43)
		SOM-BBB21	KK(Lys-P)K(AHX)( DF)CF( DW)KTC-Thr(ol)(SEQ ID NO：44)
SOM-BBB22	KKK(Lys-P)K(AHX)(AHX)( DF)CF( DW)KTC-Thr(ol)(SEQ ID NO：45)
		SOM-BBB23	(Lys-P)KK(Lys-P)K(AHX)( DF)CF( DW)KTC-Thr(ol)(SEQ ID NO：46)
SOM-BBB24	KK(Lys-P)K(AHX)KK(Lys-P)K(AHX)( DF)CF( DW)KTC-Thr(ol)(SEQ ID NO：47)
		SOM-BBB25	(PFHA)K(DK)K(ACPA)KK(Lys-P)K(AHX)( DF)CF( DW)KTC-Thr(ol)(SEQ ID NO：48)

In last table, (AHX) be that aminocaproic acid, (Dab) are N for Terleu, (Cl-Phe) for 4-chlorophenylalanine, (NN3APG) for diaminopropionic acid, (Tle) for DAB, (Dap), N-two (3-aminopropyl) glycine, (AHX) are that aminocaproic acid, (Lys-P) are Soviet Union's ammonia alcohol for Lys-palmityl, Thr (ol); _DK, _DF, _DW represents the D-isomer; PFHA is 2H, 2H, and 3H, 3H-perfluor caproic acid and ACPA are the 8-aminocaprylic acid.

Other examples that also have the analog of operable non-galanin and somatostatin.For example, δ-sleep inducing peptide (DSIP) analog is as follows:

DSIP-BBB8：(AHX)GGWAGGDASGE(SEQ ID NO：55)。Other DSIP peptides can find in table 31.

2. embodiment 2: anticonvulsant galanin analog

Galanin is a kind of 30 amino acid whose neuropeptides, compares N-terminal with full-length peptide and partly remains very effective agonist (Langel and Bartfai, 1998).Use a kind of galanin (1-16) analog (following) of truncate to introduce the modification that can strengthen its blood-brain barrier permeability.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro His Ala Val

Differentiated following for the vital residue of biological activity: Gly ¹, Trp ², Asn ⁵, Tyr ⁹And Gly ¹²(Land et al., 1991).Described N-terminal extends or the truncate meeting causes bioactive forfeiture.On the other hand, when the C-terminal of galanin (1-16) part by truncate or when being connected on the bigger structure, its can highly stable (robust) (Pooga et al., 1998).

Based on obtainable structure-activity relationship data, design, chemosynthesis and tested two kinds of galanin analog based on peptide.The structure of two kinds of analog (GAL-BBB1 and GAL-BBB2) is as follows: GAL-BBB1:Sar-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-His-(Lys-palm)-Tle-NH ₂

GAL-BBB2：Sar-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Lys-Lys-(Lys-palm)-Lys-NH ₂

Wherein Sar be sarcosine, Tle for uncle-leucine and Lys-palm for by ε amino group and the link coupled lysine residue of palmityl part, and-NH2 is illustrated in the C-terminal amidatioon.The Fmoc chemical method that described peptide uses standard is synthetic and utilize in addition purification of HPLC on solid support.Test the analog of described purification then with the epilepsy mouse model of Frings AGS susceptible.The result and the natural galanin fragment (1-16) of this research are compared.

Other analog comprise following analog:

GAL-BBB3：WTLNSAGYLLGPKKXK-NH2(SEQ ID NO：49)

GAL-BBB4：Sar-WTLNSAGYLLGP(D-Lys)(D-Lys)X(D-Lys)-NH2(SEQ ID NO：50)

GAL-BBB5：Sar-WTLNSAGYLLGPRRXR-NH2(SEQ ID NO：51)

GAL-BBB6：Sar-WTLNSAGYLLGPHHXH-NH2(SEQ ID NO：52)

GAL-BBB7：Sar-WTLNSAGYLLKKKKXK-NH2(SEQ ID NO：53)

GAL-BBB8：Sar-WTLNSAGYLLKKXK-NH2(SEQ ID NO：54)

Wherein Sar is a sarcosine, and X is a Lys-palmityl residue.

Give Frings AGS susceptible mice group with the dosage of 4mg/kg through intraperitoneal with the galanin analog of described two kinds of modifications.Different (promptly 15,30,60,120 and 240 minutes) constantly after administration place every mice in the cylinder test chamber that is equipped with audio sensor and use high strength sound stimulates (110dB, 11KHz continue 20 seconds) to attack.Think that the animal that does not show tetanic property hind leg stretching, extension is to be subjected to protection.Sum up as Figure 12, the result of this research has proved that GAL-BBB2 has the time dependence anticonvulsant action, described onset of effect time fast (in 30 minutes) and persistent period moderate (between two to four hours).On the contrary, other modified galanin analog GAL-BBB1 all do not have activity at arbitrary time point of being tested, even do not have activity when the higher dosage of 12mg/kg yet.In follow-up study, in the metered dose response studies during peak action convulsion of (1 hour) render a service.The result of this research shows that GAL-BBB2 demonstrates dosage dependence effect at AGS.Median effective dose that is calculated (being ED50) and 95% confidence interval obtain by the dose response data being carried out probit analysis, and described dosage is 3.2 (2.3-6.1) mg/kg.Described native peptides fragment GAL (1-16) is being non-activity (Figure 20) when carrying out peritoneal injection with the dosage of 20mg/kg (GAL-BBB2 ED50 6 times).

When peritoneal injection, galanin analog GAL-BBB2 demonstrates effective anti-convulsant activity (ED50 is about 3mg/kg).This Proof of Concept analog has been represented a kind of prototype, can design more " medicine sample " analog according to this prototype.Therefore can obtain to have the galanin analog of the minimum of the most effective and long lasting anti-convulsant activity.This needs a kind of two-stage process: (1) definite GAL-BBB2 analog fragment that can keep the minimum of anti-convulsant activity: this will comprise terminal and central truncate, and (2) optimize the C-terminal structural motif of the BBB permeability that will further improve described analog.At first screen synthetic analog with the galanin competitive binding assay.Use described analog to replace galanin, it is 1 μ M or the anti-convulsant activity when lower in concentration that the epilepsy mouse model by AGS further screens above-mentioned analog.Measuring those analog that when the single dose (intraperitoneal gives 2mg/kg) shown long lasting anti-convulsant activity with more pharmacology further assesses.Summed up overall experimental technique among Figure 13.Figure 23 and 24 shows respectively when giving GAL-BBB2, at the intravital time dependence anti-convulsant activity of Frings mice with in the dose dependent protective effect of the intravital anti-AGS epilepsy of Frings mice.

Carried out limited structure-functional relationship research and differentiated the minimum GAL-BBB2 analog fragment that when being given, can keep anti-convulsant activity by whole body.Synthetic and test comprises the galanin analog of C-terminal and central truncate.In addition, the C-terminal motif is carried out limited structure-functional relationship research to optimize the blood-brain barrier permeability of described analog.Figure 14 shows the GAL-BBB2 structure in structure-functional study.

Be the GAL-BBB2 analog of generation truncate, (sum up the table 11) from the Pro13 residue to the Leu10 residue and carried out 4 conservatives.Because Tyr9 is most important for the galanin activity, further the C-terminal truncate can cause biological activity to completely lose (list of references Land et al, Int J PeptProt 1991).In the analog of every kind of truncate, C-terminal " Lys-Lys-LysP-Lys " has kept the BBB permeability that improves.

Table 11The structure of GAL-BBB2 and truncate analog

For making GAL-BBB2 analog minimum, between Key residues, introduce central truncate.This is a kind of alternative approach that is used to design the bioactive peptide analog with simplified structure, can not have influence on the length of given peptide simultaneously.Main chain displacement (i.e. " main chain prosthese ") can realize by replacing two or more conservatives " non-key " residue with non-peptide sept (for example aminovaleric acid or aminocaproic acid (" main chain sept ")).This notion has better explanation in Figure 15.

With the GAL-BBB2 analog is example, by three parts (table 11) of using main chain spacer system displacement residue to detect this peptide: between Trp2 and the Asn5, between Asn5 and the Tyr9 and between Tyr9 and the C-terminal motif.Synthesize about 14 kinds of these class analog, and tested they and the combining of described galanin receptors.If some analog have kept anti-convulsant activity, then can introduce two or more septs (referring to the example in the table 12) at diverse location.

Table 12:" main chain prosthese passage (walk) " in the GAL-BBBB2 analog.Use two residues of non-peptide main chain sept (for example aminovaleric acid or aminocaproic acid) displacement can make whole molecular size reduce to minimum each time, can obviously not change the interval between the described crucial pharmacophore residue simultaneously.

Next, can optimize described C-terminal structural motif---" Lys-Lys-LysP-Lys-NH2 "---to improve the BBB permeability.Can optimize initial compounds, GAL-BBB2 by introducing following structural change (summing up in the table 13).Use homo-Lys, D-Lys or DAB displacement Lys residue to detect the BBB infiltration efficient of positive electricity residue, the side chain of described positive electricity residue has the lipophilic character of variation.Use 2-amino-tetradecanoic acid or 3, the Lys-palmityl part that the displacement of 3-two phenylalanine is 16 can determine how this position is suitable for other hydrophobic residues, and described hydrophobic residue also can strengthen the BBB permeability.

The modification of the terminal motif of table 13:C can strengthen the BBB permeability of described galanin analog.

TDA is that 2-amino-tetradecanoic acid, DAB are that DAB, D-Lys are that D isomer and the h-Lys of Lys is that homo-Lys, DPA are 3,3-two phenylalanine.

Except that the tabulation of above-mentioned analog, can produce two kinds and have the analog that lipophilic non-peptide extends at C-terminal, as follows:

Wherein X represents: 12-amino-dodecanoic acid or 2-amino-tetradecanoic acid.

As shown in figure 13, test with regard to the associativity of itself and galanin receptors every kind of synthetic and analog purification of verifying.Only replaced the analog of total length galanin further to study to those, the concentration of described analog is 1 μ M or lower.For example, galanin is implemented by NovascreenBiosciences Corporation in conjunction with measuring.Rat forebrain film and radiolabeled parental generation neuropeptide can be used in the said determination.With single concentration (1 μ M) described analog is tested to distinguish the analog of high-affinity and low-affinity.The agonist activity of selected galanin analog can use the functional examination that is provided by MDS-Pharma Services further to test.For example, can test described analog with single concentration (1 μ M).

Following table has been summed up various SAR analog, the protective effect percent that is provided by the dosage of the 4mg/kg of peritoneal injection in the time of 1,2 and 4 hour.

Table 14:

Anti-convulsant activity at first can be determined in the Frings of AGS susceptible reflex epilepsy mouse model.AGS susceptible mice is ideal acute epilepsy model because it be indifference and can effectively detect various CNS reactive compounds (White et al., 1992).Assess have active peptide in Frings mice body with regard to its ability that prevents epilepsy, described epilepsy is to utilize maximal electroshock and subcutaneous to give pentylenetetrazole (PTZ) inductive.Epilepsy chemical compound prevention epilepsy diffusion of being studied and the ability (White et al., 2002) that improves the epilepsy threshold value have been measured in above-mentioned two kinds of tests respectively.In case proved that with one or more of above-mentioned three kinds of epilepsies test a kind of modified peptide has activity, then the peak effect time after the i.p. administration of formerly determining is carried out complete dose response study.The effect research that carries out after result that above-mentioned Proof of Concept is studied and Intraventricular (i.c.v.) administration compares then.I.p. moving to left in the dose response curve can think to have realized that higher blood brain barrier penetrates.The result of above-mentioned three kinds of epilepsy test provides important data jointly, and described data have been supported the described method that makes the easier arrival brain of little peptide after the whole body administration.Hereinafter describe every kind of independent epilepsy test in detail.

Give the neuropeptide analogEach the modified neuroactive peptide that uses 10 μ l Hamilton syringes will be dissolved in the artificial cerebrospinal fluid of 5 μ l gives through Intraventricular (i.c.v.), and each modified neuroactive peptide that perhaps will be dissolved in 0.5% methylcellulose gives every kind of modified galanin analog according to every g body weight 0.01ml through intraperitoneal (i.p.).

AGSWhen peak effect, assess various modified analog prevent AGS in the Frings of AGS susceptible mouse model ability (White et al., 1992).For carrying out this test, each mice placed be equipped with audio sensor (Model AS-ZC; FETResearch and Development, Salt Lake City, (diameter 15cm in lucite tube UT); And be exposed under the sonic stimulation of 110 decibels (11KHz) continuing 20 seconds high 18cm).AGS is characterised in that bolts, and is the righting reflex forfeiture with forelimb and THE then.Think that the mice that does not show THE is to be subjected to protection.

The MES testFor carrying out the MES test, before placing Corneal electrode, an anesthesia/electrolyte solution (being dissolved in 0.9% brinish 0.5% tetracaine hydrochloride) is added drop-wise in the eye of every animal.Electricity irritation in the mice MES test is for utilizing the 50mA that continues 0.2 second a kind of and equipment output like the initial described equipment class of Woodbury and Davenport (Woodbury andDavenport, 1952).With the disappearance of the tetanic property of the hind leg of epilepsy extensor component as terminal point.

Subcutaneous PTZ testFor carrying out subcutaneous PTZ test, be that the PTZ of 85mg/kg is administered in the lax pleat skin of every mouse back through subcutaneous with dosage.Mice will be placed in the observation box of each lucite and observe in continuous 30 minutes whether have minimum clonic epilepsy outbreak.Think that the mice that does not show the clonic epilepsy seizure activity is to be subjected to protection.

Minimum toxotestTo utilize swingle method (Dunham and Miya, 1957) in the mice body, to differentiate minimum toxicity.When mice being placed speed is on 1 inch groove rasp bar of 6r.p.m. the time, and this animal can be kept its balance for a long time.If described animal fell three times from this swingle, think that then it is poisoned in 1 minute.

Determine median effective dose (ED ₅₀ ) or half toxic dose (TD ₅₀ )Formerly the TPE of Que Dinging carries out convulsion/toxicity research in all quantitative bodies.Containing each groups of at least 8 mices with the test of the peptide of various dosage, is minimum toxic boundary between establishment at least two points until the protection that is minimum toxicity and 0% in 100% protection.Utilization is calculated drug dose (ED required when producing required end points in every kind of test in 50% animal body based on the computer program of the described method of Finney (Finney, 1971) ₅₀Or TD ₅₀), 95% confidence interval, the slope of the regression line and the S.E.M. of slope.

3. embodiment 3:GAL-BBB2 has effective pain relief effect

A) formalin test

Formalin in the plantar region of the right back pawl of mice injection 0.5%.This has brought out a kind of visibly different two-phase sexual behaviour pattern, and described pattern is characterised in that mice can lick ill pawl.After the injection, mice licks pawl immediately and reaches about 10 minutes.This is the 1st phase (acute), is an of short duration incubation period afterwards, does not almost have behavioral activity this moment.Be again that a longer pawl phase of licking of about 20 to 30 minutes, this constituted the 2nd phase (inflammatory) then.

Before giving described bioactive peptide, medicine or carrier, every mice in one of high lucite observing tube of several 6 ＂ (diameter 4 ＂) has all experienced a trained reflex phase of 15 minutes, before described observing tube is placed on mirror.After phase, handle mice at described trained reflex, then it is put back into it and raises in the pipe with peritoneal injection GAL-BBB2 (the natural fragment Gal 1-16 of non-activity) or gabapentin.Handle after one hour, the formaldehyde percutaneous is injected (20 μ l down; No. 27 metering syringe needles) in right back sufficient plantar surface.The inclined-plane of syringe needle is downwards over against skin surface.After injection formaldehyde, every the mice of observation in 2 minutes per 5 minute period amounts to lasting 45 minutes.Measure the accumulated time of licking pawl in per 2 minute period.Animal and every mice that is given GAL-BBB2, Gal 1-16 or gabapentin of accepting the carrier of necessary volume replace.After this experiment finishes, put to death animal.

In further testing (in the following table), find that two kinds of other galanin analog and NAX 5055 with unique texture motif (being NAX 306-3 and 306-4) are virtuous in the mice formaldehyde determination of inflammatory pain.Above-mentioned discovery shows that described active pharmacophore is suitable for carrying out structural modification.

Table 15:

NAX	Structure	Activity is arranged
			5055	(Sar)WTLNSAGYLLGPKK(Lys-P)K(SEQ ID NO：56)	5mg/kg (" plateau II ")
306-3	(Sar)WTLNSAGYKK(Lys-P)K(SEQ IDNO：66)	(2.7mg/kg " plateau II ")
			306-4	(Sar)WTLNSAGY(Ahx)KK(Lys-P)K(SEQ ID NO：67)	(2.9mg/kg " plateau II ")

The zone of using GraphPad Prism Version 3.03 to measure under the curve (AUC).Calculate total AUC of the acute stage and the inflammatory phase of described test group and matched group.Also calculate the AUC of each issue of each animal and be converted into the percent of total AUC of accounting for contrast.Calculate average percentage and the SEM and the test significant difference of drug treating group and matched group.

B) sciatic ligation

Just before undergoing surgery, the long-acting opiate buprenorphine with 0.1 to 0.5mg/kg carries out subcutaneous treatment to rat.With pentobarbital rat is anaesthetized then and monitor depth of anesthesia to the reaction of pinching tail with to the observation of respiratory depth by them.In the whole surgery process, use aseptic technique.

Scrape hair and with ethanol and povidone iodine wiping for the thigh of every rat.Do a little otch on described skin, the leg muscle of cutting the face of wearing is until exposing sciatic nerve.Should separate and utilize thin crooked tweezers to mention gently with connective tissue on every side by nerve then.Then will be near 1/3 to 1/2 nerve knotting by pass this nerve with pin and nylon suture (7.0).With 5.0 stitching thread muscle and skin incision are sewed up respectively respectively then, and by this animal is placed into the thermostatic control heating blanket come to its warming until they from anesthesia recover.(homonymy) carries out this operation on the right side, implements sham-operation (offside) simultaneously on left back lower limb.The latter comprises a similar operation, except a sciatic nerve with this side comes out and not ligation.Operation back 12 hours, the buprenorphine that gives another dosage is to reduce any because the discomfort that this operation technique causes.Closely monitor rat with regard to the development of infection or the untoward reaction of operation every day.

Through one period suitable recovery time (for example 7-14 days) afterwards, with regard to the development of the mechanicalness pain sensation super quick (in response to the pain of non-noxious stimulation) described animal is tested.For carrying out this research, described animal is placed the lucite box at the no end, described box is positioned on silk screen (1/4 ＂) platform.Through after 30-60 minute laundering period, determine the mechanical sensitivity on basis.Vertically be put on the plantar surface of every rear solid end by VonFrey fiber and be placed on this and locate about 6 seconds (with the crooked gently described fiber of enough power) finishes this operation a series of calibrations.Recording positive reaction (this foot of withdrawing) afterwards, apply the littler fiber of diameter.Repeat this operation until the withdrawal threshold value that can determine 50%.

Behind peritoneal injection 2mg/kg GAL-BBB2 (n=8 rat/every kind of medicine), back 30 minutes of injection with and subsequent the described mechanical threshold value of different (for example 1,2,4 and 6 hours) constantly assessments, to determine the acting duration of described test compounds.To compare with result and those results who uses 2mg/kg morphine and 40mg/kg gabapentin to obtain that GAL-BBB2 obtains.

C) result

Verified many anticonvulsants can effectively be treated pain.Therefore, whether has the pain relieving characteristic with mice formaldehyde model testing GAL-BBB2 to assess it.In this test, find that GAL-BBB2 can significantly reduce and the relevant pain of s.c. vola formaldehyde, this can lick the time that the homonymy pawl spent by quantitative animal and be assessed.As shown in figure 16, during the inflammation phase of initial acute stage and prolongation GAL-BBB2 (0.52-5mg/kg) but dose dependent reduces licks foot.On the contrary, find that the natural fragment Gal-16 of unmodified after the peritoneal injection administration is a non-activity, its dosage is 4 times (being 20mg/kg) of the GAL-BBB2 maximum dose level tested.In addition, find that the GAL-BBB2 (Figure 16) of 5mg/kg is equivalent to the gabapentin (Figure 17) of 10mg/kg dosage.

As shown in figure 18, in the sciatic nerve ligation model of chronic pain, GAL-BBB2 makes the super quick threshold value of the mechanicalness pain sensation demonstrate the increase of time dependence.In addition, GAL-BBB2 is equivalent to morphine and than the high several times of effectiveness (illustration among Figure 18) of gabapentin in this test.

The result who obtains with the above-mentioned two kinds pain models of having set up has proved that jointly GAL-BBB2 has effective pain relief effect in the rodent model of chronic pain.

4. embodiment 4: the galanin analog of penetrable blood brain barrier

Table 16 shows the galanin analog that can be used for compositions disclosed herein and method:

(Sar)WTLNSAGY(D-Lys)(D-Lys)(Lys-P)(D-Lys)(SEQ ID NO：119)	Gal-BBB25
		(Sar)WTLNSAGY(Ahx)(D-Lys)(D-Lys)(Lys-P)(D-Lys)(SEQID NO：120)	Gal-BBB26
(Sar)WTLNSAGY(7-Ahp)(D-Lys)(D-Lys)(Lys-P)(D-Lys)(SEQID NO：121)	Gal-BBB27

(Sar) WTLNSAGY (3,5-two bromo-Tyr) LLGPKK (Lys-P) K (SEQ ID NO:122)	Gal-BBB28
		(Sar)WTLNSAGYLLGPHH(Lys-P)K(SEQ ID NO：123)	Gal-BBB29
(Sar)WTLNSAGYLLGPKK(Cys-Mmt)K(SEQ ID NO：124)	Gal-BBB30
		(Sar) WTLNSAGYLLGPKK (Lys-biotin-caproyl) K (SEQ ID NO:125)	Gal-BBB31
(Sar) WTLNSAGYLLGPKK (Lys-sterin) K (SEQ ID NO:126)	Gal-BBB32
		(Sar) WTLNSAGYLLGPKK (Lys-capryl) K (SEQ ID NO:127)	Gal-BBB33
(Sar) WTLNSAGYLLGPKK (Lys-caprylyl) K (SEQ ID NO:128)	Gal-BBB34
		(Sar)WTLNSAGYLLGPKK(Lys-linoyl)K(SEQ ID NO：129)	Gal-BBB35
(Sar)WTLNSAGYLLGPKK(Ser-melbiose)K(SEQ ID NO：130)	Gal-BBB36
		(Sar)WTLNSAGYLLGPKK(Lys-adamentoyl)K(SEQ ID NO：131)	Gal-BBB37
(Sar)WTLNSAGYLLGPKK(Glu(β-Lac-PEG 3-amine) K (SEQ ID NO:132)	Gal-BBB38
		(Sar) WTLTSAGYLLGPKK (Lys-palmityl) K (SEQ ID NO:133)	Gal-BBB39
(Sar) WTLLSAGYLLGPKK (Lys-palmityl) K (SEQ ID NO:134)	Gal-BBB40
		(Sar) WTLDSAGYLLGPKK (Lys-palmityl) K (SEQ ID NO:135)	Gal-BBB41

Lipotropy and alkalescence help to strengthen the blood-brain barrier permeability of peptide, and do not need unitransport albumen or carrier.The lipophilic characteristic of peptide (utilizing the logP value to measure) can be by puting together with hydrophobic part (for example lipoamino acid) or aromatic moieties being carried out halogenation transform.About alkalescence, Poduslo group has proved that albumen and peptide that polyamines is modified can more effectively pass blood brain barrier (Poduslo and Curran, 1996; Poduslo and Curran 1996; Poduslo, Curran et al 1998; Poduslo, Curran et al 1999).Tamai and colleague thereof (Tamai, Sai et al.1997) provide such evidence, the alkalescence of the increase of promptly little peptide, and to be transport protein come an important determiner by BBB via the endocytosis (AME) that absorbs mediation.

Seemingly a kind of very effective production of glycosylation has method (Elmagbari, the Egleton et al.2004 of systemically active opioid peptide; Polt, Dhanasekaran et al.2005).SAR studies show that the structure of sugar is an active important determiner, but the effectiveness of monosaccharide is usually less than disaccharide.The 0-glycosylation serine that contains β-6-(.alpha.-D-galactosido)-D-glucose. or beta lactose all is the most effective modifications that can produce effective analgesic compounds.

The effect in epilepsy and epilepsy invasion of galanin and receptor thereof.Neuropeptide is effectively modified (Hokfelt, the Broberger et al.2000) of typical neurotransmitter and neuronal excitability.Neuropeptide and the coexistence of typical neurotransmitter in selected neuron pool mean to conduct by the modification Toplink regulates neuronal excitability (Baraban and Tallent 2004).Around under the condition, peptide is " silence " and can exert an influence to normal neurotransmission hardly.On the contrary, under the very excited situation of neuron (as occurring in the epilepsy kitchen range), neuropeptide can be released and neurotransmission is produced regulating action.

Galanin produces multiple effect (Hokfelt, Xu et al.1998 in brain; Lundstrom, Elmquist et al.2005).Identify three kinds of galanin receptors hypotypes at present and belong to g protein coupled receptor superfamily (GPCR) (Branchek, Smith et al.2000; Lundstrom, Elmquist etal.2005).1 type galanin receptors (GalR1) exists in many brains zone, but shows the highest expression (Burgevin, Loquet et al.1995) in Hippocampus.The distribution of 2 type galanin receptors (GalR2) is equally extensive with GalR1.In brain, it is expressed in hypothalamus, Hippocampus (dentate gyrus〉CA3〉CA1), corpus amygdaloideum, PC, basal forebrain (medial septal nuclear/diagonal bundle), cerebellum and brain stem.3 type galanin receptors (GalR3) show very limited expression in brain.It is very abundant in hypothalamus, medial reticular formation and diagonal bundle, but is not present in the Hippocampus.

Because Mazarati and colleague's thereof pioneering work (Mazarati, Halaszi et al.1992) exists more and more evidences to confirm that galanin is a kind of effective convulsion peptide.Have been found that the acute administration of galanin receptors agonist or virus-mediated galanin cross that expression can suppress rat and the intravital edge of mice is epilepsy state, pentylenetetrazole and Picrotoxin epilepsy (Mazarati, Halaszi et al.1992 in Hippocampus; Mazarati, Liu et al.1998; Saar, Mazarati et al.2002; Haberman, Samul ski et al.2003; Lin, Richichiet al.2003; Bartfai, Lu et al.2004).In addition, raise (Mazarati, Hohmannet al.2000 of the epilepsy threshold value that galanin is crossed the express transgenic animal in status epilepticus and kindling model; Kokaia, Holmberg et al.2001; Schlifke, Kuteeva et al.2006).

External, galanin can suppress Hippocampus release glutamate (Zini, Roisin et al.1993; Mazarati, Hohmann et al.2000).The result of the research of using the GalR1 knock-out mice and carrying out with GalR2 peptide antisensenucleic acids shows that galanin is brought into play its anticonvulsant action (Mazarati, Lu et al.2004 by acting on GalR1 and GalR2 simultaneously; Mazarati, Lu et al.2004).In addition, think GalR2 in the neuroprotective of galanin, play an important role (Haberman et al., 2003 to hippocampal neuron; Mazarati et al., 2004a; Pirondi et al., 2005; Elliot-Hunt et al., 2004; Lee et al., 2005; Hwang et al., 2004).

Should be emphasized that galanin can effectively prevent the expression of acute epilepsy and alleviate the epilepsy development afterwards of various wounds.For example, several pieces of reports have proved that galanin can alleviate the damage relevant with the edge epilepsy and delay or prevent the development (being epilepsy) of epilepsy.Kokaia etc. (Kokaia, Holmberg et al 2001) have reported that crossing the intravital delay of expression mice at galanin lights.Recently the result of a research of carrying out in kindling model fast shows that the Hippocampus GalR2 with the Gi/o albumen coupling can not rely on GIRK and brings into play antiepileptic action, GIRK activates light (Mazarati, the Lundstrom et al.2006) that causes yet GalR1 can postpone.

Structure-activity-relation (SAR) in galanin and the galanin receptors part.Galanin is found (Tatemoto, Rokaeus et al 1983) first in nineteen eighty-three.It is the peptide of a kind of 29-30 amino acid long, has following sequence:

The people

AVGNHRSFSDKNGLTS- _COOH(SEQ ID NO:93)

Rat/mouse

AIDNHRSFSDKHGLT- _NH2(SEQ ID NO:94)

Pig

AIDNHRSFHDKYGLA- _NH2(SEQ ID NO:95)

14 residues of initial N-terminal (shade) are high conservative (Langel and Bartfai 1998) in the galanin sequence of different animals species.Because the structure-activity-relation of galanin has obtained extensive studies, so we will only look back those SAR results relevant with the application.---being made up of 15 initial residues (analog GAL (1-15)) or 16 residues (analog GAL (1-16))---can keep high-affinity (Fisone, Berthold et al.1989 to galanin receptors to have proved the N-terminal fragment of GAL; Land, Langel et al.1991).As shown in the table, system's truncate meeting of GAL (1-16) is caused the gradually reduction (Land, Langel et al 1991) of GAL (1-16) to the affinity of its receptor.

Table 17: to the influence of the segmental truncate of GAL (1-16) to the affinity of it and galanin receptors

Fragment	Sequence	K D[μM]
			1-16	GWTLNSAGYLLGPHAI(SEQ ID NO：96)	0.007
1-14	GWTLNSAGYLLGPH(SEQ IDNO：97)	0.15
			1-12	GWTLNSAGYLLG (SEQ ID NO：98)	3
1-10	GWTLNSAGYL (SEQ ID NO：99)	25
			1-9	GWTLNSAGY (SEQ ID NO：100)	100

In identical research, the author has proved that Gly1, Trp2, Asn5, Tyr9 and Gly12 residue combine very important for GAL (1-16) analog with the high-affinity of galanin receptors.The alanine passage analog of GAL (1-16) shows that the displacement of above-mentioned residue can influence them to GalR2 but not the affinity of GalR1 (Carpenter, Schmidt et al.1999).The alanine method of elimination shows that the sequence of crossing over Try9 to His14 is for the identification of galanin receptors most important (Jureus, Langel et al.1997).

(Pooga, Jureus et al.1998) described the SAR research very widely that GAL (1-16) analog is carried out.The modification of Gly1 or Trp2 is caused obvious forfeiture to the affinity of galanin receptors.All also have GAL (1-13) analog of Lys14 ε-NH group to keep high-affinity, the different group couplings of described NH group and size variation.According to The above results, the C-terminal of GAL part can be held huge relatively group, and can not influence in conjunction with character.In a word, the SAR that galanin and truncate analog thereof are carried out show Trp2, Asn5 and Tyr9 be used to protect galanin and GalR1 and and the GarR2 receptor between interactional Key residues.Mutation and Modeling Research show that Trp2 can interact with the Phe282 of hGalR1, and Tyr9 and His264 interaction (Kask et al, 1996; Berthold et al 1997; Church et al, 2002).

Because structure-activity relation has synthesized and the functional many galanin analog based on peptide (agonist and antagonist) that characterized widely.Summing up of table 18 pair selected galanin receptors part in conjunction with character.

The selected galanin receptors part of table 18 and with different galanin receptors hypotype in conjunction with character

^aK _DValue is collected from (Branchek, Smith et al.2000; Lundstrom, Elmquist etal.2005; Lundstrom, Sollenberg et al.2005; Sollenberg, Lundstromet al.2006).Galnon and galmic are the galanin receptors agonist of two kinds of non-peptides, and they have become and are used for studying very useful pharmacological tool (Saar, the Mazarati et al.2002 of galanin receptors in the effect of CNS; Bartfai, Lu et al.2004; Badie-Mahdavi, Behrens et al.2005; Lu, Barr et al.2005; Schlifke, Kuteeva et al.2006).Yet, state as nearest: " shortcoming of Galnon and galmic is that they are (micromole's affinity) of low affinity, no receptor subtype selectivity and the target interaction upward important with other pharmacologys ... " (Lu, Lundstrom et al.2005).

Appropriate design and the chemosynthesis of NAX5055 (the galanin analog of a kind of penetrable BBB).As disclosed herein, galanin is a kind of 30 amino acid whose neuropeptides, and verified N-terminal fragment GAL (1-16) remains a kind of to the effective agonist of Hippocampus galanin receptors height (Fisone, Berthold et al.1989).At present the result who obtains is to use (Figure 31) that the analog GAL (1-16) of truncate obtains, and described analog has carried out modified by a kind of mode that increases metabolic stability and heighten blood-brain barrier permeability.

According to obtainable structure-activity relation data, introduce following modification to improve its metabolic stability and blood-brain barrier permeability in GAL (1-16) analog: (1) Gly1 residue is replaced by sarcosine.The N of Gly1 methylates can not influence galanin receptors affinity (Rivera Baeza, Kask et al.1994).Therefore in addition, known Aminopeptidase N degradable neuropeptide may reduce metabolic degradation speed to the analog that contains sarcosine to the capping of N-terminal amino group; (2) His14 and Ala15 are replaced by the Lys residue.Add amidatioon Lys residue at C-terminal.Above-mentioned extra positive electricity can strengthen by the BBB permeability that endocytosis mediated (Tamai, Sai et al.1997) that absorbs mediation; (3) Val16 is replaced by lysine-palmityl (Lys-palm) residue.This long hydrophobic residue can increase passive diffusion and extra metabolic degradation resistance (Yuan, Wang et al.2005) is provided.NAX 5055 is to use the Fmoc scheme of standard and automatic peptide synthesizer chemically synthetic on solid support.

The pharmacological property of NAX 5055 has utilized radioligand in conjunction with measuring and the epilepsy outbreak of having to go to the toilet of one group of body is tested NAX 5055 is assessed up to now.It is modified that the result who has above-mentioned research to obtain shows that method disclosed herein can produce effective high-affinity galanin receptors, described modified penetrable BBB.

NAX 5055 has kept utilizing preliminary radioligand in conjunction with the affinity that studies confirm that 5055 couples of people hGalR1 of NAX and hGalR2 to the high-affinity of GalR1 and GalR2, described research is carried out (report#1077561, reference: MDSPS231510 and 231600) by MDS-PS contract screening company.In this research, hGalR1 HEK-293 cellular expression, and hGalR2 CHO-K1 cellular expression, and the end user [ ¹²⁵I] galanin is as radioligand.NAX 5055 has kept the high-affinity to two kinds of hypotypes, estimates that Ki to hGalR1 is 9nM and is 6nM to the Ki of hGalR2.

NAX 5055 demonstrates effective anti-convulsant activity after intraperitoneal gives 4mg/kg after the whole body administration, the Frings reflex epilepsy mouse model of using AGS (AGS) susceptible is to NAX

5055 carry out initial testing.Different (promptly 15,30,60,120,120 and 240 minutes) constantly after administration place every mice in the cylinder test chamber that is equipped with audio sensor and use high strength sound stimulates (110dB, 11KHz continue 20 seconds) to attack.

Think that the animal that does not show tetanic property hind leg stretching, extension has been subjected to protection as shown in figure 24; this result of experiment has proved that NAX5055 has the time dependence anticonvulsant action, described onset of effect time fast (in 30 minutes) and persistent period moderate (between two to four hours).In follow-up dose response study, the quantitative convulsion effectiveness of (1 hour) during peak action.Median effective dose that is calculated (being ED50) and 95% confidence interval obtain by the dose response data being carried out probit analysis, and described dosage is 3.2 (2.3-6.1) mg/kg.When 1 hour when test after the intraperitoneal administration, NAX 5055 (4mg/kg) and non-natural GAL (1-16) fragment (20mg/kg) has effectively stoped AGS in Frings mice body.

Also NAX 5055 is tested: i.e. maximal electroshock seizure (the tetanic clonic spasm epilepsy model of general) and subcutaneous pentetrazole epilepsy test (general myoclonus epilepsy model) with two kinds of perfect epilepsy models.For carrying out this research, give 4mg/kg NAX 5055 through intraperitoneal, and as a child mice was tested with regard to the protective effect of anti-tonic extension (maximal electroshock) and clonicity (subcutaneous pentetrazole) epilepsy 1.NAX 5055 be lowest activity (25% protective effect) in the test of subcutaneous pentetrazole epilepsy, and in the maximal electroshock seizure test complete non-activity (result is not shown).Although above-mentioned result can be interpreted as negative findings from the angle of clinical potency, but the distribution of described up to now NAX 5055 was in fact identical with novel antiepileptic levetiracetam, and described levetiracetam was introduced into the partial seizures that is used for the treatment of the people in 2000.

Therefore, in the process of the anticonvulsant action of making great efforts enhancing NAX 5055, we also use the acute epilepsy model of another kind of levetiracetam sensitivity (being the test of 6Hz psychomotor) that it is assessed.Described 6Hz epilepsy test is developing into a kind of unique model that is used to distinguish potential convulsion chemical compound, and described chemical compound can be used for treating intractable partial epilepsy (Barton, Klein etal.2001; White 2003).In the model of this drug resistant epilepsy, NAX 5055 very effectively (table 19) after the intraperitoneal administration.Different with levetiracetam even valproic acid, when stimulus intensity increased to 44mA by 22, tiring of NAX 5055 obtained maintenance.Therefore, NAX 5055 is unique relatively in the anticonvulsant drug of 6Hz test test, because it is still very effective under whole three kinds of assessment current intensity.On the contrary, when stimulus intensity increased to 44mA by 22, tiring of other drug all decreased.Subsequently with mice 6Hz limbic lobe outbreak model measurement the NAX5055 after subcutaneous (s.c.) administration.Activity has obtained maintenance (Figure 26) after it should be noted that subcutaneous administration.The ED50 of NAX has only taken place slightly to move to right behind the subcutaneous administration, and this discovery shows that NAX 5055 has good bioavailability.

When making great efforts to confirm that described native peptides fragment enters in the brain is in the activated process, has carried out a follow-up study, wherein NAX 5055 and GAL (1-16) all directly is administered in the ventricles of the brain space.Result to this Intraventricular research among Figure 22 sums up.As shown in figure 22, two kinds of analog after the Intraventricular administration all very effectively (be NAX 5055 and ED ₅₀Be respectively 0.07 and 1.7 nanomoles).It should be noted that NAX 5055 tire and usefulness may be all apparently higher than described native peptides fragment GAL (1-16).

Table 19

The structure-activity relation of NAX 5055 analog.After the whole body administration, carried out SAR research, purpose is to understand its active structures determiner better.At first, tested with the combination of two kinds of modifications and compared, whether independent C-terminal chemical modification can produce similar influence.Shown in hereinafter, to compare with NAX 5055, independent modification does not have permanent effectively anti-convulsant activity.Analog 1105-2 demonstrates lower tiring and (compares ED with 5055 0.8mg/kg ₅₀=3.8mg/kg), short action time and observed toxicity (dyskinesia) in 5055 not.The existence of Lys-palm residue is not enough to produce any observable anti-convulsant activity.The above results is intensive to have hinted that the combination of two kinds of modifications is extremely important for the pharmacological property of 5055 analog.

Table 20

Analog	Structure		1, the protective effect percent that provides by the dosage of 4 mg/kg of lumbar injection 2 and 4 hours the time
				Gal(1-16)	GWTLNSAGYLLGPHAV(SEQ ID NO：1)	Non-activity
5055	(Sar)WTLNSAGYLLGPKK(Lys-P)K(SEQ ID NO：56)	100％，100％，0％
			1105-2	(Sar)WTLNSAGYLLGPKKKK(SEQ IDNO：60)	30%, 0%, 0%, poison
306-5	(Sar)WTLNSAGYLLGPHA(Lys-P)(SEQ ID NO：68)	Non-activity

Annotate: all analog comprise that NAX 5055 has all carried out amidatioon at C-terminal.

Secondly, by using _DLys or Arg replace the Lys residue or study the effect of the Lys residue that is positioned at C-terminal by changing many Lys residues.Use Arg displacement Lys only to change the activity of described analog a little.Use the Lys isomer. _DLys displacement Lys can produce one and have the similar (ED that tires ₅₀=1-2mg/kg) long-acting analog (1205-2).Can keep anti-convulsant activity with two extra Lys residue displacement Gly12-Pro13, but also can toxigenicity.21 pairs of above-mentioned result of experiment of following table are summed up:

NAX	Structure		1, the protective effect percent that provides by the dosage of 4 mg/kg of lumbar injection 2 and 4 hours the time
				5055	(Sar)WTLNSAGYLLGPKK(Lys-P)K(SEQ ID NO：56)	100％，100％，0％
1205-2	(Sar)WTLNSAGYLLGPDKDK(Lys-P)DK (SEQ ID NO：69)	100％，50％，75％
			1205-3	(Sar)WTLNSAGYLLGPRR(Lys-P)R(SEQ ID NO：70)	100％，75％，0％
1205-4	(Sar)WTLNSAGYLLKKKK(Lys-P)K(SEQ ID NO：71)	75%, 100%, 66%, poison

Next, determined the influence of the central truncate of NAX 5055 to function.Shown in table 22, " G12, the P13 " of Gal (1-16) or " L10, L11, G12, P13 " truncate meeting are with its at least two order of magnitude of affinity reduction to galanin receptors.In the 6Hz model, the systemic central truncate meeting of analog 5055 (analog 1205-5 and 306-3) produces tiring of only reducing a little and (compares the ED of two kinds of analog with the 0.8mg/kg of analog 5055 ₅₀Be 2.7mg/kg) and short action time.

Table 22

NAX	Structure		1, the protective effect percent that provides by the dosage of 4 mg/kg of lumbar injection 2 and 4 hours the time
				5055	(Sar)WTLNSAGYLLGPKK(Lys-P)K(SEQID NO：56)	100％，100％，0％
1205-5	(Sar)WTLNSAGYLLKK(Lys-P)K(SEQ IDNO：72)	100％，25％，0％
			306-3	(Sar)WTLNSAGYKK(Lys-P)K(SEQ IDNO：66)	75％，25％，0％

Introduce main chain sept (for example 6-aminocaprolc acid) between galanin fragment and KKKpK motif, described introducing may influence the anti-convulsant activity of described analog.Analog 306-2 and 306-4 can keep its anti-convulsant activity (ED of 306-4 ₅₀=2.95mg/kg).It should be noted that, when dosage is 2.95mg/kg (ED50 in the 6Hz model), as if second of the inflammatory pain mensuration that analog 306-4 carries out in the mice body is replied interim very effective, shows that tiring of its anti-convulsant activity and analgesic activities may not be directly related.

Table 23

At last, detected the activity (analog 1205-1:WTLNSAGYLLGPKK (Lys-P) K) of the analog 5055 of disappearance N-terminal sarcosine residue.This analog designs according to galanin agonist analog Gal (2-11), described Gal (2-11) is called AR-M1896 again: supposition removes first residue and will reduce the affinity of described analog to the GalR1 hypotype, keeps the high-affinity (referring to the binding data of Gal in the table 21 (2-11) analog) to GalR2 simultaneously.Analog 1205-1 has the (ED that tires of reduction in 6Hz limbic lobe epilepsy model ₅₀=5.7mg/kg).

In a word, SAR result shows that the combination of cationization and fatization is better than independent chemical modification.

A) design and the chemosynthesis of the galanin analog of penetrable BBB

According to existing SAR result, synthetic and sign contains the analog of chemical modification combination.Adopted a kind of new cationization method: use chemical compound (for example spermine) or fat-polyamines conjugate based on polyamines.Because the glycosylation of peptide is a kind of maturation method that improves the BBB permeability of peptide, so adopted glycosylated galanin analog.

CationizationThe combination of positive electricity Lys residue and they and Lys-palm can improve described analog sending in the CNS.The example of above-mentioned conservative analog relatively is shown in the table 24.

Table 24 has the analog that the KKKpK motif is modified.All analog all at C-terminal by amidatioon.

Analog	Principle
		(Sar) WTLNSAGYLLGPKK (Lys-P) K (SEQ ID NO:56)	NAX 5055 (as with reference to illustrating)
(Sar) (Om) (Lys-P) (Om) (SEQ ID NO:73) of WTLNSAGYLLGP (Om)	Use ornithine displacement Lys can produce the shorter side chain of a few methylene.This has played corresponding hydrophobic interaction in the BBB of NAX 5055 infiltration
		(Sar) (Dab) (Lys-P) (Dab) (SEQ ID NO:74) of WTLNSAGYLLGP (Dab)	Can produce the shorter side chain of few two methylene with 2,4-diamino-butanoic (Dab) displacement Lys.Referring to above-mentioned principle.
(Sar) WTLNSAGYLLGPbKbK (Lys-P) bK (SEQ ID NO:75)	β-Gao-Lys be (1) metabolic stability and (2) more hydrophobic.
		(Sar) WTLNSAGYLLGPHH (Lys-P) H (SEQ ID NO:76)	Can reduce the alkalescence of described analog with His displacement Lys
(Sar) WTLNSAGYLLGP (Lys-P) KKK (SEQ ID NO:77)	Change the relative position of Lys and Lys-palm residue
		(Sar) WTLNSAGYLLGPK (Lys-P) KK (SEQ ID NO:78)	The same
(Sar) WTLNSAGYLLGPKKK (Lys-P) (SEQ ID NO:79)	The same

Because cationization is penetrating most important for the blood brain barrier of galanin analog, so studied the galanin analog that contains polyamines.The principle that use contains the analog of spermine is to use individual molecule to replace several Lys residues (being present among the NAX 5055), described individual molecule carries several amino groups, but lacks peptide bond (proteoclastic susceptibility is reduced and lacks hydrogen bond donor/receptor).Synthesized several galanin analog, wherein spermine is a part or the side chain of main chain.Table 25 has been summed up the structure and the principle of galanin-spermine analog.

Table 25 contains spermine as the main chain sept or as NAX 5055 analog of side chain.Spermine S is 1,5,10, the 14-four azepines tetradecane-N4-succinic acid.All analog all at C-terminal by amidatioon.

Analog	Principle
		(Sar) WTLNSAGY (spermine S) (Lys-P) (SEQ ID NO:80)	Spermine-N4 succinic acid has 18 backbone atoms, can replace 6 AA in principle.Spermine S can be used as " LLGPKK " among the basic at interval displacement NAX 5055 of main chain.
(Sar) WTLNSAGYLLGPKK (Lys-P)-(spermine S) (SEQ ID NO:81)	To increase the alkalescence of described analog with spermine displacement Lys17.
		(Sar) WTLNSAGYLLGPKK (Glu-spermine) K (SEQ ID NO:82)	With replacing Lys-palm with the spermine of glutamic acid side chain coupling
(Sar) WTLNSAGYLLGPKK (Lys-spermine-palmityl) K (SEQ ID NO:83)	To increase the alkalescence of described analog with spermine-palmityl displacement Lys-palm part.

FatizationLike (table 26) in the thing, utilized of the influence of the 16th fatization at next category to the BBB infiltration of compositions.

Table 26 has the analog of the 16th modification.All analog all at C-terminal by amidatioon.

Analog	Principle
		(Sar) WTLNSAGYLLGPKK (TDA) K (SEQ ID NO:84)	Short lipoamino acid is used in test: the effect the when tetradecane is partly replaced Lys-palmityl part
(Sar) WTLNSAGYLLGPKK (NorL) K (SEQ ID NO:85)	Use nor-leucine displacement Lys-palmityl part can produce a kind of palmityl residue (and amino group) " truncate " analog that lacked.

GlycosylationAt first synthesize two kinds at the 16th glycosylated galanin analog that contains α-mannose group or β-6-(.alpha.-D-galactosido)-D-glucose. serine residue.Therefore, in above-mentioned analog, sugar moieties has been replaced the Lys-palm residue.

The glycosylated galanin analog of table 27.All analog all at C-terminal by amidatioon.

Analog	Principle
		(Sar) WTLNSAGYLLGPKK (Lys-P) K (SEQ ID NO:56)	NAX 5055 (as with reference to illustrating)
(Sar) WTLNSAGYLLGPKK (Man) K (SEQ ID NO:86)	Monosaccharide derivatives is used in test: the influence when L-Ser-α-mannose is replaced Lys-palmityl part.
		(Sar) WTLNSAGYLLGPKK (Mel) K (SEQ ID NO:87)	Two sugar derivativess are used in test: the influence when L-Ser-β-6-(.alpha.-D-galactosido)-D-glucose. is replaced Lys-palmityl part.Principle is the same

The main chain septStudied of the influence of main chain sept to the anti-convulsant activity of NAX 5055 basic analog.Main advantage with the peptide main chain of non-sept replacing section based on peptide is: (1) reduces molecular size, and this can improve the BBB permeability, and susceptibility and (3) that (2) reduce proteolytic degradation lack hydrogen bond donor/receptor.The example of above-mentioned analog is shown in the table 28.

Table 28 contains NAX 5055 analog of the glycine main chain sept of prolongation.All analog all at C-terminal by amidatioon.

Analog	Sept
		(Sar)WTLNSAGYLL(1PEG)KK(Lys-P)K(SEQID NO：88)	5-amino-3-oxopentanoic acid
(Sar)WTLNSAGYLL(5AVA)KK(Lys-P)K(SEQID NO：89)	The 5-aminovaleric acid
		(Sar)WTLNSAGYL(2PEG)KK(Lys-P)K(SEQ IDNO：90)	8-amino-3, the 6-dioxo is sad
(Sar)WTLNSAGYL(8AOA)KK(Lys-P)K(SEQID NO：91)	The 8-aminocaprylic acid
		(Sar)WTLNSAGY(1PEG)(5AVA)KK(Lys-P)K(SEQ ID NO：92)	5-amino-3-oxopentanoic acid, 5-aminovaleric acid

The chemosynthesis of described analogAll analog all can use synthetic (SPPS) scheme of Fmoc base-solid phase-peptide and automatic peptide synthesizer to synthesize.All SPPS building blocks all are commercially available, comprise Fmoc protection main chain sept, glycoprotein amino acid, spermine, spermine-succinic acid or spermine-palmityl (Sussex Research Laboratories, Iris Biotech, NeoMPS, Chem-Impex).Will be according to previous (Fields and Noble 1990; Albericio 2000) described enforcement coupling method.At the 16th analog of puting together the spermine or derivatives thereof, described analog has been introduced the glutamic acid with γ-2-benzene isopropyl esters (0-2-PhiPr) protection for wherein.The 1%TFA that use is dissolved in dichloromethane on resin with the side chain carboxyl group deprotection.The coupling of spermine or its palmityl derivant (Fmoc/Boc protects other amino groups) is to use 1, and 3-DIC (DIC) is carried out.Described peptide is shifted out, washes and precipitate with MTBE from solid support.Described analog uses diphenyl to prepare type reversed-phase HPLC purification.Use acetonitrile (being dissolved in 0.1% the TFA) (eluting of from 20% to 90% 90% acetonitrile/10% water/0.1%TFA) 15 minutes of linear gradient.The molar absorption coefficient 7,000 at use 280nm place is determined the amount (1 Tpr 5,600 and 1 Tyr 1,400) of analog.Said method can effectively prepare NAX5055 and similar analog, and each batch scope is the 10-50 microgram.

B) can pass the vitro characterization of the galanin analog of BBB

By characterizing the biological activity of synthetic galanin analog, determined the binding constant of GalR1 and GalR2 receptor.Three main targets are: (1) assessment chemical modification that to be what factor affecting be incorporated among the GAL (1-16) changes the affinity to galanin receptors, (2) determine the selectivity distribution of galanin analog and a kind of reliable Screening test method of (3) exploitation can be passed BBB with screening high-affinity galanin analog.Verified NAX5055 has kept the GalR1 and the GalR2 affinity of low nanomole level.

Can use a kind of binding assay based on fluorescence (Delfia of PerkinElmer measures) that uses the galanin of europium labelling.This mensuration---described---through checking by (Valenzano, Miller et al.2000) before and can be used for characterizing all previous synthetic analog in conjunction with character.Compare with radioligand assay commonly used more, described main advantage in conjunction with measuring based on fluorescence be can avoid radioactive shortcoming (be harmful to health, not easy to clean, the pot-life is short, the picked up signal required time is long), make this class measure and be more suitable in middle flux and high flux screening.Be hypersensitivity with the available advantage of the receptors ligand of lanthanum labelling, because they have long fluorescence lifetime.The time-resolved fluorescence that use to postpone 400 microseconds detects, and can detect to be lower than 1 europium that flies mole in single hole.

The galanin receptors of two kinds of film preparation forms is available from Perkin Elmer or Multispan, Inc..The galanin of europium labelling (Eu-Galanin) is available from Perkin Elmer.Association reaction is to use the 10 microgram memebrane proteins (concentration) that are dissolved in 60 microlitre binding buffer liquid (being dissolved in EDTA, BSA, the PEG of hypotonic buffer liquid) to carry out.Using the ligand concentration scope is that 0.01 to 10nM Eu-Galanin forms saturated binding curve.For determining binding constant, Eu-Galanin and the film of 0.2nM were hatched 2 hours.Come cessation reaction by using vacuum box to filter the AeroWell filter plate fast, and wash three times with 300mL hypotonic buffer liquid.To strengthen solution joins in each hole and with TFR write down the TRF signal on the Victor3 spectrofluorimeter.

C) can pass in the body of galanin analog of BBB and characterize

Based on the neuropeptide of galanin in the drug resistance model of epilepsy anti-convulsant activity and their epilepsy character.

6Hz limbic lobe epilepsy model with drug resistance part epilepsy characterizes selected analog.Determine tiring of analog described in this drug resistant epilepsy model by the dose response curve after the administration of generation intraperitoneal.All chemical compounds are the intraperitoneal administration or are dissolved in 0.9% NaCl and come administration according to every g body weight 0.01ml.Carry out the acute effect research except that testing, also analyzed NAX 5055 and other galanin analog prevent to light acquisition in the mice cornea kindling model of partial epilepsy ability (Matagne and Klitgaard 1998) with the 6Hz epilepsy.

The convulsion test.Prevent the ability of epilepsy with every kind of analog of three kinds of different stimulus intensities (promptly 22,32 and 44mA) assessment, described epilepsy stimulates (continuing 3 seconds) to bring out by the 6Hz cornea.Described 6Hz epilepsy is mechanical spontaneous behaviour after being characterised in that the minimum clonic spasm phase, and described behavior is considered to and the aura similar (Toman, the Everett et al.1952 that suffer from the human patients of partial seizures at first; Barton, Klein et al.2001).Think that the animal that does not show the behavior all is to be subjected to protection.As indicated above, when electric current increased to 44mA by 22mA, described 6Hz epilepsy was stronger to the resistance of the blocking-up of antiepileptic.According to (Barton, Klein etal.2001) described method, quantize the activity of every kind of analog during peak action after the intraperitoneal administration with the test of 6Hz epilepsy.For carrying out this test, before placing Corneal electrode, an anesthesia/electrolyte solution (being dissolved in 0.9% brinish 0.5% tetracaine hydrochloride) is added drop-wise in the eye of every animal.For carrying out the peak action time study, 20 CF-1 mices have been used altogether.In the difference moment (promptly 0.25,0.5,1,2 and 4 hour) 4 mices of each group are tested.For carrying out dose response study, contain each groups of at least 8 mices with candidate's peptide test of various dosage, until at least two points of establishment between 100% usefulness and 0% the usefulness.Utilization is calculated drug dose (ED required when producing half effect dosage in 50% the animal body that is contacted based on the computer program of the described method of Finney (Finney, 1971) ₅₀), 95% confidence interval, the slope of the regression line and the S.E.M. of slope.

The acquisition cornea is lightedDescribed according to (Matagne and Klitgaard 1998), other galanin analog of assessment NAX 5055 and selected quantity prevent the ability that obtains to light in mice cornea kindling model.Corneal was carried out the electricity irritation meeting produced the stable state of lighting in 15-20 day every day.This is a kind ofly to be used for the ideal model that the selected galanin analog of initial assessment prevents the ability that obtains to light, because the amount of required peptide is lighted the amount of the peptide that institute needs far below carrying out rat.Assess those with rat kindling model (being the corpus amygdaloideum kindled rats) commonly used more subsequently and can effectively prevent the chemical compound that obtains to light.For carrying out described research, two groups of mices of picked at random (n=8 mice/every group) were accepted carrier or peptide at every turn before lighting stimulation.With the approaching ED that is used to prevent 6Hz (44mA) epilepsy ₅₀Dosage give every kind of peptide through intraperitoneal.In the moment of peak action (available from described 6Hz research), all mices all stimulate (50Hz, 3mA last 3 sec) with Corneal electrode with electric current under the convulsant threshold, and observe whether there is seizure activity.According to Racine et al., the standard that (1972) are established is marked to seizure activity.Animal is accepted twice stimulation every day, shows the 5th grade of behavior epilepsy first until them, stimulates once every day then, shows stable secondary generalized seizures (5 seriality 4-5 level epilepsy) until them.In described experimental group, proceed the peptide treatment, lighted fully until described control mice.At this moment, the mice in two groups has all been accepted one-week stimulation and has not been had treatment week.During by the 8th day, under the situation that does not have peptide or carrier, two groups mice stimulated and writes down their epilepsy scoring.

When finishing to the second stimulation week, the mice of vehicle treated group can reach the state of lighting fully (Figure 32 and 33).In addition, consider to have been found that before the i.c.v. galanin can prevent to light development (Mazarati, Lundstrom et al 2006) that the mice of described experimental group can not lighted or be lighted with the speed that is slower than the vehicle treated mice.Therefore, the epilepsy scoring that described activity is lighted the last day of research is 1 or still less, and behind described ejection time (wash-out period), described epilepsy scoring is still very low; (epilepsy) for example handled.If the epilepsy behind the ejection time of described experimental group scoring and vehicle treated group, then can sum up the effect of described peptide during the active processing phase obviously different owing to its anticonvulsant action causes; (convulsion) for example handled.Whether when observing antiepileptic action, all mices of vehicle group and processed group are all lighted under the situation that does not have medicine, can light under the situation that does not have peptide with the mice of observing first pre-treatment.

5. embodiment 5: the design of analog and chemosynthesis

A) can pass the neuropeptide analog of BBB

Table 29 has been summed up the structure of several neuropeptide analog.For every kind of analog, described " KKK _PK " motif can be connected to N or C-terminal in the solid-phase peptide building-up process.Following table provides the brief principle that is used to design every kind of neuropeptide analog.

Table 29The structure of selected neuropeptide

Neuropeptide	Structure
		SOM	Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys(SEQ ID NO：30)
Octreotide-NH2	DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-NH 2(SEQ ID NO：101)
		DSIP	Trp-Ala-Gly-Gly-Asp-Phe-Ser-Gly-Glu(SEQ ID NO：102)
Dynorphin A (1-16)	Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln(SEQ ID NO：103)
		NPY(13-36)	PAEDLARYYSALRAYINLITRQRY-NH 2(SEQ ID NO：104)

SomatostatinCan " hold " that N-terminal extends and biological activity (the Dasgupta and Mukherjee 2000 that can not have influence on them with its subtype-selective analog---octreotide---; Dasgupta, Singh et al.2002; Na, Murty et al.2003).The N-terminal acetylation of RC-160 analog can cause the CNS concentration of this somatostatin analogs to increase (Banks, Schally et al.1990).Octreotide also can penetrate BBB to a certain extent and not need extra modification (Fricker, Nobmann et al.2002).

DSIP。Proved the antiepileptic activity that N-terminal extends can not influence DSIP, and C-terminal extends meeting generation non-activity analog.Therefore, can produce the DSIP analog by carrier being connected to N-terminal.

N-terminal at SOM, octreotide or DSIP is introduced described " KKK _P K " motifUse three kinds of different septs (Gly, 6-aminocaprolc acid (Ahx), Ahx-Gly) to connect described " KKK _PK " motif is so that the interactional probability minimum of the influence of described big Lys-palm residue and target receptor.Synthesized following analog:

KK (K _p) K-(neuropeptide) (SEQ ID NO:105)

KK (K _p) KG-(neuropeptide) (SEQ ID NO:106)

KK (K _p) K (Ahx)-(neuropeptide) (SEQ ID NO:107)

KK (K _p) K (Ahx) G-(neuropeptide) (SEQ ID NO:108)

Dynorphin A (1-16)Can be at C-terminal by truncate or modified, and can obviously not reduce affinity (Lapalu, Moisand et al.1997 to opioid receptor; Naqvi, Haq et al.1998; Schlechtingen, DeHaven et al.2003).Therefore, similar to the galanin analog of penetrable BBB, can synthesize dynorphin A (1-16) analog, wherein last several residues are by described " KKK _PK " motif replaces.Show following analog:

Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Lys-(Lys-palm)-Lys-NH ₂(SEQ ID NO：109)

Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Lys-Lys-(Lys-palm)-Lys-NH ₂(SEQ ID NO：110)

Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Lys-Lys-(Lys-palm)-Lys-NH ₂(SEQID NO：111)

Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-(Ahx)-Lys-Lys-(Lys-palm)-Lys-NH ₂(SEQ ID NO：112)

Neuropeptide tyrosineAnalog can design according to the data of many SAR researchs of carrying out in Annette G.Beck-Sickinger professor's laboratory and (comprehensively summarize (Beck-Sickinger and Jung1995; Cabrele and Beck-Sickinger 2000)).Particularly, described design is based on ^99mThe NPY analog of Tc-labelling synthesizes described analog and tests it whether can be used as tumor developer (Langer, La Bella et al.2001).The NPY analog of truncate (Ac-[Ahx5-24, K4 (99mTc (CO) 3-PADA) A26]-NPY)---contain big 2-aminomethyl-pyridine-N, the N-oxalic acid (has chelating ^99mTc)---to the very effective (IC of Y2 receptor subtype ₅₀=1nM).Therefore, 2-aminomethyl-pyridine-N, the N-oxalic acid can be replaced by Lys-palmityl residue.Show following two kinds of NPY analog:

KKK(K _p)(Ahx)RAYINLITRQRY-NH ₂(SEQ ID NO：113)

KK(K _p)K(Ahx)RAYINLITRQRY-NH ₂(SEQ ID NO：114)

Also produce 4 kinds and had NPY (13-36) analog that N-terminal extends:

KK(K _p)K-[NPY(13-36)](SEQ ID NO：115)

KK(K _p)KG-[NPY(13-36)](SEQ ID NO：116)

KK(K _p)K(Ahx)-[NPY(13-36)](SEQ ID NO：117)

KK(K _p)K(Ahx)G-[NPY(13-36)](SEQ ID NO：118)

All analog all can use synthetic (SPPS) scheme of the Fmoc base-solid phase-peptide of standard and automatic peptide synthesizer to synthesize.All SPPS building blocks all are commercially available, comprise Fmoc protection aminocaproic acid, Lys-palmityl and glycoprotein amino acid (Sussex Research Laboratories, IrisBiotech, NeoMPS, Chem-Impex).Will be according to previous (Fields and Noble 1990; Albericio 2000) described enforcement coupling method.Described peptide is shifted out, washes and precipitate with MTBE from solid support.Described analog uses diphenyl to prepare type reversed-phase HPLC purification.Use linear gradient to carry out eluting.Determine initial concentration and the final concentration of the 90% acetonitrile/10% water/0.1%TFA of every kind of analog according to the retention time of every kind of analog in analytical HPLC analyzes.Use every kind of analog to determine the amount (Tpr ε=5,600 and Tyr ε=1,400) of analog at the molar absorption coefficient at 280nm place.Said method can effectively prepare the galanin analog, and its quantitative range is the 10-50 milligram.Be the disulfide bond in oxidation somatostatin or the octreotide analog, use Clear-Ox resin (Darlak, Wiegandt Long et al.2004; Green and Bulaj 2006).Octreotide analog as herein described is carried out oxidation, and productive rate surpasses 95%.Utilize preparation HPLC purification oxidation end-product.

The anti-convulsant activity of neuropeptide analog characterizes analog with the 6Hz limbic lobe epilepsy model of drug resistance part epilepsy.Can determine the activity of described analog after i.c.v. and the i.p. administration.Described method is at first to test the analog of Intraventricular administration (2nmoles): use i.p. bolus injection (4mg/kg) further to test active analogue thereof.Also tested carry out the i.c.v. administration with 2nmoles after, the anti-convulsant activity of dynorphin A (1-16), NPY and NPY (13-36).Above-mentioned reference value as its modified analog of screening.

The convulsion test is assessed the ability that every kind of analog prevents epilepsy with the stimulus intensity of 32mA, and described epilepsy stimulates (continuing 3 seconds) to bring out by the 6Hz cornea.Described 6Hz epilepsy is mechanical spontaneous behaviour after being characterised in that the minimum clonic spasm phase, and described behavior is considered to and the aura similar (Toman, the Everett et al.1952 that suffer from the human patients of partial seizures at first; Barton, Klein et al.2001).Think that the animal that does not show the behavior all is to be subjected to protection.According to (Barton, Klein et al.2001) described method, quantize the activity of every kind of analog during peak action after Intraventricular or intraperitoneal administration with the test of 6Hz epilepsy.For carrying out this test, before placing Corneal electrode, an anesthesia/electrolyte solution (being dissolved in 0.9% brinish 0.5% tetracaine hydrochloride) is added drop-wise in the eye of every animal.For carrying out the peak action time study, 20 CF-1 mices have been used altogether.Difference constantly (promptly 0.25,0.5 .1,2 and 4 hours) 4 mices of each group are tested.All chemical compounds all are dissolved in 0.9% NaCl and come administration according to every g body weight 0.01ml.

As if the design of glycosylation neuropeptide analog and chemosynthesis glycosylation can improve BBB infiltration (Elmagbari, the Egleton et al.2004 of opioid peptide very effectively; Polt, Dhanasekaranet al.2005).Figure 34 shows the structure of the saccharide residue that can be used for ICI154129 N,N-bisallyl-Tyr-Gly-Gly-.β-6-(.alpha.-D-galactosido)-D-glucose. is incorporated in the described peptide, after the i.v. administration, can produces analog with best analgesic effect.

β-6-(.alpha.-D-galactosido)-D-glucose .-Ser residue be directed in the selected neuropeptide analog to replace total length " KKK _PK " motif or Lys-palmityl residue (as indicated above).For the described glycosylation analog of chemosynthesis, used full acetyl group-β-6-(.alpha.-D-galactosido)-D-glucose .-Ser derivant available from the Fmoc protection of Sussex Research.Used the solid phase synthesis scheme of standard.The deacetylation of 6-(.alpha.-D-galactosido)-D-glucose. residue can be by realizing with the hatching of (pH 10) of pH regulation and control of in the 10mM Feldalat NM that is melted into methanol analog being carried out 4-5 hour.Described analog utilizes preparation HPLC to come purification, and uses the traditional vacuum drying system that it is carried out drying before its anti-convulsant activity of test.

6. embodiment 6:NAX5055 lights the influence of acquisition to the mice cornea

With the CF#1 mice study blood brain barrier penetrating agent---light the ability of development based on the neuropeptide NAX-5055 of galanin prevention cornea.Mice cornea kindling model is a kind of non-invasive partial epilepsy animal model, and electric current (3mA) under the mice initial convulsant threshold that continues 3 seconds having accepted to be provided by Corneal electrode wherein continues a few days twice every day.The mice of vehicle treated needs to reach over 12-16 days usually stablizes the 5th grade of epilepsy; It is Secondary cases whole body focus epilepsy.In this research, 16 mices are divided into two experimental grouies at random: saline group or NAX-5055 group.Mice in the NAX-5055 group they be subjected to before cornea stimulates first 12 hours and 1 hour and accepted in 1 hour before the subsequent stimuli at every turn peritoneal injection NAX-5055 (4mg/kg, n=8).On the contrary, the NaCl (n=8) of the acceptance 0.9% in 1 hour before each cornea stimulates of the mice in the vehicle group.Before each the stimulation, be dissolved in 0.9% brinish 0.5% tetracaine with one and be added drop-wise on the cornea.After the stimulation, seizure activity is marked according to the 0-5 grade standard that Racine (1972) is established.Proceed treatment and show the 5th grade of epilepsy all the time until control animal.

Shown in Figure 32 and 33, the mice in the NAX-5055 group is divided into two independent groups: promptly to those mices (n=3) of NAX-5055 treatment sensitivity with to NAX-5055 treatment insensitive those mices (n=5).Sensitivity is defined as those mices that in stimulating course, can't show the 5th grade of epilepsy.Compare with the insensitive animal of NAX-5055 with contrast, the responsive animal of NAX-5055 needs relatively large stimulation to reach the 1st, the 2nd and 3rd level.In addition, compare with the insensitive animal of NAX-5055 with contrast, in the one-week stimulation of experience with when not having peptide and attacking once more the phase after, the sizable stimulation of the responsive animal needs of NAX-5055 reach the 4/5th grade of epilepsy.The above results shows that NAX-5055 can postpone to light to obtain, and therefore can be used for the patient is carried out the early treatment, described patient easily suffers from epilepsy (Racine RJ.Modification of seizure activity byelectrical stimulation.II.Motor seizure.Electroencephalogr ClinNeurophys iol.1972 Mar after being subjected to brain injury; 32 (3): 281-94.).

Table 30 shows octreotide or the anti-convulsant activity of DSIP analog after the whole body administration.

Analog	Protective effect percent in the time of 30 minutes (4mg/kg, i.p.)
		Octreotide
DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-NH 2	50％
		Lys-Lys-Lys(palm)-Lys-Ahx-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-NH 2	100％
The DSIP analog
		Trp-Ala-Gly-Gly-Asp-Phe-Ser-Gly-Glu	0％
Ahx-Gly-Gly-Trp-Ala-Gly-Gly-Asp-Phe-Ser-Gly-Glu	50% (after 2 hours 100%)

Table 31 shows other δ-sleep inducing peptide:

Analog	Structure
		DSIP-BBB8	(Ahx)GGWAGGDASGE(SEQ ID NO：136)
DSIP-BBB99	(Palm)GGWAGGDASGE(SEQ ID NO：137)
		DSIP-BBB100	(K p)GGWAGGDASGE(SEQ ID NO：138)
DSIP-BBB101	KKK(K p)GGWAGGDASGE(SEQ ID NO：139)
		DSIP-BBB102	KK(K p)KGGWAGGDASGE(SEQ ID NO：140)
DSIP-BBB103	KKKGGWAGGDASGE(SEQ ID NO：141)
		DSIP-BBB104	(DK)(DK)(DK)(Kp)GGWAGGDASGE(SEQ ID NO：58)

G. sequence

SEQ ID NO:1 (wild type galanin)

Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro His Ala Val

SEQ ID NO：2(GAL-BBB1)

Sar-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-His-(Lys-palm)-Tle-NH2

(wherein Sar be sarcosine, Tle for uncle-leucine and Lys-palm for by ε amino group and the link coupled lysine residue of palmityl part, and-NH2 is illustrated in the C-terminal amidatioon)

SEQ ID NO：3(GAL-BBB2)

Sar-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Lys-Lys-(Lys-palm)-Lys-NH2

(wherein Sar be sarcosine, Tle for uncle-leucine and Lys-palm for by ε amino group and the link coupled lysine residue of palmityl part, and-NH2 is illustrated in the C-terminal amidatioon)

SEQ ID NO:4 (from table 11)

Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu：

Gly Pro Lys Lys Lys-P Lys

SEQ ID NO:5 (from table 11)

Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu

Gly Lys Lys Lys-P Lys

SEQ ID NO:6 (from table 11)

Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu

Lys Lys Lys-P Lys

SEQ ID NO:7 (from table 11)

Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Lys

Lys Lys-P Lys

SEQ ID NO:8 (from table 11)

Sar Trp Thr Leu Asn Ser Ala Gly Tyr Lys Lys

Lys-P Lys

SEQ ID NO:9 (from table 12)

Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu

Gly Pro Lys Lys Lys-P Lys

SEQ ID NO:10 (from table 12)

Sar Trp Xaa Xaa Asn Ser Ala Gly Tyr Leu Leu

Gly Pro Lys Lys Lys-P Lys

SEQ ID NO:11 (from table 12)

Sar Trp Thr Leu Asn Xaa Xaa Gly Tyr Leu Leu

Gly Pro Lys Lys Lys-P Lys

SEQ ID NO:1 2 (from table 12)

Sar Trp Thr Leu Asn Ser Xaa Xaa Tyr Leu Leu

Gly Pro Lys Lys Lys-P Lys

SEQ ID NO:13 (from table 12)

Sar Trp Thr Leu Asn Ser Ala Gly Tyr Xaa Xaa

Gly Pro Lys Lys Lys-P Lys

SEQ ID NO:14 (from table 12)

Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Xaa

Xaa Pro Lys Lys Lys-P Lys

SEQ ID NO:15 (from table 12)

Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu

Xaa Xaa Lys Lys Lys-P Lys

SEQ ID NO:16 (from table 12)

Sar Trp Thr Leu Asn Xaa Xaa Xaa Tyr Leu Leu

Gly Pro Lys Lys Lys-P Lys

SEQ ID NO:17 (from table 12)

Sar Trp Xaa Xaa Asn Xaa Xaa Xaa Tyr Leu Leu

Gly Pro Lys Lys Lys-P Lys

((SEQ ID NOS:9-17) " Xaa " represents a sept and can be any length in the sequence of table 12)

SEQ ID NO:18 (from table 13)

Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu

Gly Pro Lys Lys Lys-P Lys

SEQ ID NO:19 (from table 13)

Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu

Gly Lys Lys Lys Lys-P Lys

SEQ ID NO:20 (from table 13)

Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu

Lys Lys Lys Lys Lys-P Lys

SEQ ID NO:21 (from table 13)

Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu

Gly Pro Lys Lys Lys Lys

SEQ ID NO:22 (from table 13)

Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu

Gly Pro Lys Lys-P Lys Lys

SEQ ID NO:23 (from table 13)

Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu

Gly Pro Lys-P Lys Lys Lys

SEQ ID NO:24 (from table 13)

Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu

Gly Pro D-Lys D-Lys Lys-P D-Lys

SEQ ID NO:25 (from table 13)

Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu

Gly Pro h-Lys h-Lys Lys-P h-Lys

SEQ ID NO:26 (from table 13)

Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu

Gly Pro DAB DAB Lys-P DAB

SEQ ID NO:27 (from table 13)

Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu

Gly Pro Lys Lys TDA Lys

SEQ ID NO:28 (from table 13)

Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu

Gly Pro Lys Lys DPA Lys

In above-mentioned table 3 sequence (SEQ ID NOS:18-28), TDA is the amino tetradecylic acid of 2-, and DAB is a DAB, and D-Lys is that D-isomer and the h-Lys of Lys is height-Lys, and DPA is 3,3-two phenylalanine

SEQ ID NO:29 (from table 13)

Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu

Gly Pro Lys Lys Lys-P Lys X

Wherein X represents: 12-amino-dodecoic acid or 2-amino-tetradecylic acid

SEQ ID NO:30 (wild type somatostatin)

Ala Gly Cys Lys Asn Phe Phe Trp Lys Thr Phe

Thr Ser Cys

SEQ ID NO:31 (from table 5)

Ala Ala Cys Lys DAB Phe Phe D-Trp Lys DAP Phe

DAP DAP Cys

SEQ ID NO:32 (from table 5)

Ala Gly Cys Lys-P Lys-P Phe Phe D-Trp Lys Thr Phe

Thr Ser Cys

SEQ ID NO:33 (from table 5)

Ala Gly Cys Lys Asn Cl-Phe Cl-Phe D-Trp Lys Thr Cl-Phe

Thr Ser Cys

SEQ ID NO:34 (from table 5)

Ala Ala Cys Lys DAB Phe Phe L-Trp Lys DAP Phe

DAP DAP Cys

SEQ ID NO:35 (from table 5)

Ala Gly Cys Lys-P Lys-P Phe Phe L-Trp Lys Thr Phe

Thr Ser Cys

SEQ ID NO：36(From Table 5)

Ala Gly Cys Lys Asn Cl-Phe Cl-Phe L-Trp Lys Thr Cl-Phe

Thr Ser Cys

In the sequence of above-mentioned table 5, DAB is a DAB; DAP is a diaminopropionic acid; Lys-palm is the Lys-palmityl; And Cl-Phe is chloro-Phe.

SEQ ID NO:37 (from table 9)

Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu

Gly Lys-p Lys-p Lys-p Val

SEQ ID NO:38 (from table 9)

Sar Trp DAP Leu Asn DAP DAP Gly Tyr Leu Leu

Gly DAB His DAP DAB

SEQ ID NO:39 (from table 9)

Sar Trp Thr Leu Asn Ser Ala Gly Cl-TyrLeu Leu

Gly Pro His Ala Val

SEQ ID NO:40 (octreotide that has modification)

D-Phe Cys Phe D-Trp Lys Thr Cys Thr(ol)

SEQ ID NO：41(SOM-BBB1)

(NN3APG)(AHX)AGCKNFFWKTFTSC

SEQ ID NO：42(SOM-BBB2)

(NN3APG)(AHX)AGCKNFF(DW)KT(Cl-Phe)T(Dap)C

SEQ ID NO：43(SOM-BBB3)

W(AHX)KKCKNFF(DW)KT(Cl-Phe)(Dab)(Dab)C

SEQ ID NO：44(SOM-BBB21)

KK(Lys-P)K(AHX)(DF)CF(DW)KTC-Thr(ol)

SEQ ID NO：45(SOM-BBB22)

KKK(Lys-P)K(AHX)(AHX)(DF)CF(DW)KTC-Thr(ol)

SEQ ID NO：46(SOM-BBB23)

(Lys-P)KK(Lys-P)K(AHX)(DF)CF(DW)KTC-Thr(ol)

SEQ ID NO：47(SOM-BBB24)

KK(Lys-P)K(AHX)KK(Lys-P)K(AHX)(DF)CF(DW)KTC-Thr(ol)

SEQ ID NO：48(SOM-BBB25)

(PFHA)K(DK)K(ACPA)KK(Lys-P)K(AHX)(DF)CF(DW)KTC-Thr(ol)

For SEQ ID NOS:41-48, (AHX) be aminocaproic acid; (Dab)=DAB;

(Dap)=diaminopropionic acid; (Tle)=uncle-leucine; (Cl-Phe)=the 4-chlorophenylalanine;

(NN3APG)=and N, N-two (3-aminopropyl) glycine; (AHX)=aminocaproic acid; (Lys-P)=the Lys-palmityl;

Thr (ol)=Soviet Union's ammonia alcohol; DK, DF, DW represent the D-isomer; PFHA is 2H, 2H, 3H, 3H-perfluor caproic acid; And ACPA is the 8-aminocaprylic acid

SEQ ID NO：49 GAL-BBB3

WTLNSAGYLLGPKKXK-NH2

SEQ ID NO：50 GAL-BBB4

Sar-WTLNSAGYLLGP(D-Lys)(D-Lys)X(D-Lys)-NH ₂

SEQ ID NO：51 GAL-BBB5

Sar-WTLNSAGYLLGPRRXR-NH2

SEQ ID NO：52 GAL-BBB6

Sar-WTLNSAGYLLGPHHXH-NH2

SEQ ID NO：53 GAL-BBB7

Sar-WTLNSAGYLLKKKKXK-NH2

SEQ ID NO：54 GAL-BBB8

Sar-WTLNSAGYLLKKXK-NH2

In SEQ ID NOS:49-54, Sar is that sarcosine and X are Lys-palmityl residue

SEQ ID NO：55 DSIP-BBB8

(AHX)GGWAGGDAS GE

SEQ ID NO：56

(Sar)WTLNSAGYLLGPKK(Lys-P)K

SEQ ID NO：57

WTLNSAGYLLGPKK(Lys-P)K

SEQ ID NO：58

(DK)(DK)(DK)(Kp)GGWAGGDASGE

SEQ ID NO：59

(Sar)WTLNSAGYLLGPRR(Lys-P)R

SEQ ID NO：60

(Sar)WTLNSAGYLLKKKK(Lys-P)K

SEQ ID NO：61

(Sar)WTLNSAGYLLGPKKKK

SEQ ID NO：62

(Sar)WTLNSAGYLLKK(Lys-P)K

SEQ ID NO：63

(Sar)WTLNSAGYKK(Lys-P)K

SEQ ID NO：64

(Sar)WTLNSAGYLLGP(Ahx)KK(Lys-P)K

SEQ ID NO：65

(Sar)WTLNSAGY(Ahx)KK(Lys-P)K

SEQ ID NO：66

(Sar)WTLNSAGYKK(Lys-P)K

SEQ ID NO：67

(Sar)WTLNSAGY(Ahx)KK(Lys-P)K

SEQ ID NO：68

(Sar)WTLNSAGYLLGPHA(Lys-P)

SEQ ID NO：69

(Sar)WTLNSAGYLLGP _DK _DK(Lys-P) _DK

SEQ ID NO：70

(Sar)WTLNSAGYLLGPRR(Lys-P)R

SEQ ID NO：71

(Sar)WTLNSAGYLLKKKK(Lys-P)K

SEQ ID NO：72

(Sar)WTLNSAGYLLKK(Lys-P)K

SEQ ID NO：73

(Sar)WTLNSAGYLLGP(Om)(Om)(Lys-P)(Om)

SEQ ID NO：74

(Sar)WTLNSAGYLLGP(Dab)(Dab)(Lys-P)(Dab)

SEQ ID NO：75

(Sar)WTLNSAGYLLGPbKbK(Lys-P)bK

SEQ ID NO：76

(Sar)WTLNSAGYLLGPHH(Lys-P)H

SEQ ID NO：77

(Sar)WTLNSAGYLLGP(Lys-P)KKK

SEQ ID NO：78

(Sar)WTLNSAGYLLGPK(Lys-P)KK

SEQ ID NO：79

(Sar)WTLNSAGYLLGPKKK(Lys-P)

SEQ ID NO：80

(Sar)WTLNSAGY(SpermineS)(Lys-P)

SEQ ID NO：81

(Sar) WTLNSAGYLLGPKK (Lys-P)-(spermine S)

SEQ ID NO：82

(Sar) WTLNSAGYLLGPKK (Glu-spermine) K

SEQ ID NO：83

(Sar) WTLNSAGYLLGPKK (Lys-spermine-palmityl) K

SEQ ID NO：84

(Sar)WTLNSAGYLLGPKK(TDA)K

SEQ ID NO：85

(Sar)WTLNSAGYLLGPKK(NorL)K

SEQ ID NO：86

(Sar)WTLNSAGYLLGPKK(Man)K

SEQ ID NO：87

(Sar)WTLNSAGYLLGPKK(Mel)K

SEQ ID NO：88

(Sar)WTLNSAGYLL(1PEG)KK(Lys-P)K

SEQ ID NO：89

(Sar)WTLNSAGYLL(5AVA)KK(Lys-P)K

SEQ ID NO：90

(Sar)WTLNSAGYL(2PEG)KK(Lys-P)K

SEQ ID NO：91

(Sar)WTLNSAGYL(8AOA)KK(Lys-P)K

SEQ ID NO：92

(Sar)WTLNSAGY(1PEG)(5AVA)KK(Lys-P)K

SEQ ID NO：93

AVGNHRSFSDKNGLTS- _COOH

SEQ ID NO：94

AIDNHRSFSDKHGLT- _NH2

SEQ ID NO：95

AIDNHRSFHDKYGLA- _NH2

SEQ ID NO：96

GWTLNSAGYLLGPHAI

SEQ ID NO：97

GWTLNSAGYLLGPH

SEQ ID NO：98

GWTLNSAGYLLG

SEQ ID NO：99

GWTLNSAGYL

SEQ ID NO：100

GWTLNSAGY

In above-mentioned sequence:

The Ahx=aminocaproic acid

_DThe D isomer of K=lysine

The Orn=ornithine

Dab=2, the 4-DAB

_bK=β-high-lysine

Spermine S=spermine-N ₄Succinic acid

The TDA=tetradecanoic acid

The NorL=nor-leucine

Man=L-Ser-β-6-(.alpha.-D-galactosido)-D-glucose.

1-PEG=5-amino-3-oxa-valeric acid

The 5AVA=5-aminovaleric acid

2-PEG=8-amino-3,6-two oxa-s are sad

The 8AOA=8-aminocaprylic acid

SEQ ID NO：101

_DPhe-Cys-Phe- _DTrp-Lys-Thr-Cys-Thr-NH ₂

SEQ ID NO：102

Trp-Ala-Gly-Gly-Asp-Phe-Ser-Gly-Glu

SEQ ID NO：103

Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln

SEQ ID NO：104

PAEDLARYYSALRAYINLITRQRY-NH ₂

SEQ ID NO：105

KK (Kp) KX (wherein X can be random length and arbitrary amino acid)

SEQ ID NO：106

KK (Kp) KGX (wherein X can be random length or arbitrary amino acid)

SEQ ID NO：107

KK (Kp) K (Ahx)-(neuropeptide) wherein X can be random length or arbitrary amino acid)

SEQ ID NO：108

KK (Kp) K (Ahx) G-(neuropeptide) (wherein X can be random length or arbitrary amino acid)

SEQ ID NO：109

Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Lys-(Lys-palm)-Lys-NH ₂

SEQ ID NO：110

Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Lys-Lys-(Lys-palm)-Lys-NH ₂

SEQ ID NO：111

Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Lys-Lys-(Lys-palm)-Lys-NH ₂

SEQ ID NO：112

Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-(Ahx)-Lys-Lys-(Lys-palm)-Lys-NH ₂

SEQ ID NO：113

KKK(Kp)(Ahx)RAYINLITRQRY-NH ₂

SEQ ID NO：114

KK(Kp)K(Ahx)RAYINLITRQRY-NH ₂

SEQ ID NO：115

KK (Kp) K-[X (13-36)] (wherein a kind of neuropeptide tyrosine analog of X (13-36) representative)

SEQ ID NO：116

KK (Kp) KG-[X (13-36)] (wherein a kind of neuropeptide tyrosine analog of X (13-36) representative)

SEQ ID NO：117

KK (Kp) K (Ahx)-[X (13-36)] (wherein a kind of neuropeptide tyrosine analog of X (13-36) representative)

SEQ ID NO：118

KK (Kp) K (Ahx) G-[X (13-36)] (wherein a kind of neuropeptide tyrosine analog of X (13-36) representative)

SEQ ID NO：119

(Sar)WTLNSAGY(D-Lys)(D-Lys)(Lys-P)(D-Lys)

SEQ ID NO：120

(Sar)WTLNSAGY(Ahx)(D-Lys)(D-Lys)(Lys-P)(D-Lys)

SEQ ID NO：121

(Sar)WTLNSAGY(7-Ahp)(D-Lys)(D-Lys)(Lys-P)(D-Lys)

SEQ ID NO：122

(Sar) WTLNSAGY (3,5-two bromo-Tyr) LLGPKK (Lys-P) K

SEQ ID NO：123

(Sar)WTLNSAGYLLGPHH(Lys-P)K

SEQ ID NO：124

(Sar)WTLNSAGYLLGPKK(Cys-Mmt)K

SEQ ID NO：125

(Sar) WTLNSAGYLLGPKK (Lys-biotin-ammonia caproyl) K

SEQ ID NO：126

(Sar) WTLNSAGYLLGPKK (Lys-sterin) K

SEQ ID NO：127

(Sar) WTLNSAGYLLGPKK (Lys-capryl) K

SEQ ID NO：128

(Sar) WTLNSAGYLLGPKK (Lys-caprylyl) K

SEQ ID NO：129

(Sar) WTLNSAGYLLGPKK (the inferior oleoyl of Lys-) K

SEQ ID NO：130

(Sar) WTLNSAGYLLGPKK (Ser-6-(.alpha.-D-galactosido)-D-glucose .) K

SEQ ID NO：131

(Sar) WTLNSAGYLLGPKK (Lys-diamantane (obsolete) acyl) K

SEQ ID NO：132

(Sar) WTLNSAGYLLGPKK (Glu (β-Lac-PEG3-amine)) K

SEQ ID NO：133

(Sar) WTLTSAGYLLGPKK (Lys-palmityl) K

SEQ ID NO：134

(Sar) WTLLSAGYLLGPKK (Lys-palmityl) K

SEQ ID NO：135

(Sar) WTLDSAGYLLGPKK (Lys-palmityl) K

SEQ ID NO：136

(Ahx)GGWAGGDASGE

SEQ ID NO：137

(Palm)GGWAGGDASGE

SEQ ID NO：138

(Kp)GGWAGGDASGE

SEQ ID NO：139

KKK(Kp)GGWAGGDASGE

SEQ ID NO：140

KK(Kp)KGGWAGGDASGE

SEQ ID NO：141

KKKGGWAGGDASGE

For SEQ ID NOS:58 and 135-141, (DK) be D-Lys, (K _p) be the Lys-palmityl, Axh is an aminocaproic acid, Palm is a Palmic acid

Claims (56)

1. isolating polypeptide comprises SEQ ID NO:3, has aminoacid sequence at least about 90% homogeneity with the aminoacid sequence of SEQ ID NO:3, perhaps has the aminoacid sequence of the metathetical SEQ ID of one or more conservative amino acid NO:3.

2. isolating polypeptide, comprise and be selected from SEQ ID NO:2,4-29,37-39,50,64,65,66,67,80,82 and 89 aminoacid sequence, with be selected from SEQ ID NO:2,4-29,37-39,50,64,65,66,67,80,82 and 89 aminoacid sequence has the aminoacid sequence at least about 90% homogeneity, perhaps has the metathetical SEQ of the being selected from IDNO:2 of one or more conservative amino acid, 4-29,37-39,50,64,65,66,67,80,82 and 89 aminoacid sequence.

3. isolating polypeptide, comprise the amino acid fragment that is selected from SEQ ID NO:31-36, have hydrogen base acid sequence, perhaps have the aminoacid sequence of the metathetical SEQ of the being selected from ID of one or more conservative amino acid NO:31-36 at least about 90% homogeneity with the aminoacid sequence that is selected from SEQ ID NO:31-36.

4. an isolating polypeptide comprises SEQ ID NO:40, has aminoacid sequence at least about 90% homogeneity with the aminoacid sequence of SEQ ID NO:40, or has the aminoacid sequence of the metathetical SEQ ID of one or more conservative amino acid NO:40.

5. isolating polypeptide, comprise the aminoacid sequence that is selected from SEQ ID NO:105,106,107,108,109-112 and 113-118, have aminoacid sequence with the aminoacid sequence that is selected from SEQ ID NO:105,106,107,108,109-112 and 113-118, perhaps have the aminoacid sequence of the metathetical SEQ of the being selected from ID of one or more conservative amino acid NO:105,106,107,108,109-112 and 113-118 at least about 90% homogeneity.

6. isolating polypeptide, comprise the amino acid fragment that is selected from SEQ ID NO:58 and 135-141, have aminoacid sequence with the aminoacid sequence that is selected from SEQ ID NO:58 and 135-141, perhaps have the aminoacid sequence of one or more conservative amino acid metathetical SEQ ID NO:58 and 135-141 at least about 90% homogeneity.

7. claim 1 or 2 polypeptide, wherein said polypeptide is compared with galanin has enhanced stability.

8. the polypeptide of claim 3, wherein said polypeptide is compared with somatostatin has enhanced stability.

9. the polypeptide of claim 6, wherein said polypeptide is compared with δ-sleep inducing peptide (DSIP) has enhanced stability.

10. compositions with enhanced blood-brain barrier permeability, wherein said compositions comprise a kind ofly compares the peptide with enhanced lipophilic characteristic and enhanced alkalescence with this peptide of not transformed.

11. the compositions of claim 10, other compositionss of wherein said compositions and one or more are used in combination.

12. the method for claim 10, wherein said other compositionss are selected from: neuropeptide tyrosine, dynorphin, opioid and opioid peptide, morphine, hydroxyl morphine, fentanyl, oxycodone, codeine; Capsaicin; And antiepileptic class, include but not limited to carbamazepine, primidone, gabapentin, pregabalin, diazepam, felbamate, fluoro felbamate (fluorofelbamate), lamotrigine, lacosamide, levetiracetam, phenobarbital, phenytoin, fosphenytoin, topiramate, valproate, vigabatrin, zonisamide, oxcarbazepine, non-steroidal antiinflammatory drugs (NSAID), local anesthetic (for example lignocaine), glutamate receptor antagonist, nmda antagonist, alpha-2-adrenoceptor agonist and antagonist, adenosine, Cannabinoids, NK-1 antagonist (CI-1021), antidepressants (amitriptyline for example, desipramine, imipramine), the analog of galanin and derivant, somatostatin, δ-sleep inducing peptide, enkephalin, oxytocin, Pancreozymin, calcitonin, hydrocortisone hormone inhibin (cortistatin), nociceptin and other therapeutic agent based on neuropeptide, pluronic P85 block copolymer, amyloid depressant, for example Flurizan; Galantamine; Profit is cut down this bright (Exelon); Donepezil (aricept); Tacrine (romotal); Memantine (Namenda) and other are used for the vaccine of Alzheimer.

13. each compositions of claim 10-12, wherein said lipophilic characteristic can be strengthened by described peptide and a kind of hydrophobic part are puted together.

14. the compositions of claim 10, wherein said hydrophobic part is poly-aliphatic chain.

15. each compositions of claim 10-14, wherein said lipophilic characteristic can be strengthened by the halogenation that increases aromatic moieties.

16. each compositions of claim 10-15, wherein said alkalescence can be strengthened by same oligomer and the different oligomer of introducing the positive electricity amino acid residue, and described positive electricity amino acid residue includes but not limited to the ornithine of lysine, arginine, high-lysine, homoarginine, L-or D-isomer configuration; 2, the 3-diaminopropionic acid; 2,4-diamino-butanoic.

17. each compositions of claim 10-15, wherein said alkalescence can be by being strengthened with puting together based on the part of polyamines, and described part based on polyamines is spermine, spermidine, polyamide-amide type dendrimer or polyamines toxin and derivant thereof for example.

18. each compositions of claim 10-17, wherein said peptide is compared with this peptide of not transformed, and the efficient of passing blood brain barrier can improve 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

19. each compositions of claim 10-18, wherein said peptide is compared the glycosylation that also has increase with this peptide of not transformed.

20. carrier that contains each described compositions of claim 10-18.

21. a method that increases the blood-brain barrier permeability of peptide comprises comparing when making described peptide and not transformed to have enhanced lipophilic characteristic and enhanced alkalescence.

22. the method for claim 21, wherein said lipophilic characteristic can be strengthened by described peptide and hydrophobic part are puted together.

23. the method for claim 22, wherein said hydrophobic part is poly-aliphatic chain.

24. the method for claim 21, wherein said lipophilic characteristic can be strengthened by the halogenation that increases aromatic moieties.

25. the method for claim 21, wherein said alkalescence can be strengthened by same oligomer and the different oligomer of introducing the positive electricity amino acid residue, and described positive electricity amino acid residue includes but not limited to the ornithine of lysine, arginine, high-lysine, homoarginine, L-or D-isomer configuration; 2, the 3-diaminopropionic acid; 2,4-diamino-butanoic.

26. the method for claim 21, wherein said alkalescence can be by being strengthened with puting together based on the part of polyamines, described part based on polyamines is spermine, spermidine, polyamide-amide type dendrimer or polyamines toxin and derivant thereof for example.

27. the method for claim 21, wherein said peptide is compared with this peptide of not transformed, and the efficient of passing blood brain barrier can improve 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

28. the method for claim 21 comprises that also described peptide compares with this peptide of not transformed and also have enhanced glycosylation.

29. a method for the treatment of epilepsy comprises the described polypeptide of claim 1 of experimenter's effective dose that needs treatment.

30. a method for the treatment of epilepsy comprises the described compositions of claim 10-19 of experimenter's effective dose that needs treatment.

31. the method for claim 30, wherein said epilepsy are generalized epilepsy, partial epilepsy or intractable epilepsy.

32. the method for a treatment, prevention or alleviating pain or other neurological disorder comprises the described polypeptide of claim 1 of experimenter's effective dose that needs treatment.

33. the method for claim 32, wherein said pain be cause by one or more following diseases or described mental disorder be selected from one or more following diseases: chronic back pain, cancer, fibromyalgia, postherpetic neuralgia, multiple sclerosis, diabetic neuropathy, peripheral nerve injury, traumatic mononeuropathy, complex region pain syndrome and spinal cord injury.

34. a method for the treatment of spinal cord injury in the subject comprises giving described each described polypeptide of experimenter's claim 10-12.

35. a method for the treatment of multiple sclerosis in the subject comprises giving described each described polypeptide of experimenter's claim 10-12.

36. a treatment, prevention or alleviate the method for neurological disorder comprise the described polypeptide of claim 2 that needs experimenter's effective dose for the treatment of.

37. a treatment, prevention or alleviate the method for neurological disorder comprise the described polypeptide of claim 3 that needs experimenter's effective dose for the treatment of.

38. each method of claim 32 or 36-37, wherein said neurological disorder is an epilepsy.

39. each method of claim 32 or 36-37, wherein said neurological disorder are depressed.

40. each method of claim 32 or 36-37, wherein said neurological disorder is a pain.

41. each method of claim 32 or 36-37, wherein said neurological disorder is an Alzheimer.

42. each method of claim 32 or 36-37 is wherein also used the described experimenter of one or more other combination treatments.

43. one kind is used for the treatment of experimenter's the method that needs can pass the compositions of blood brain barrier, comprises:

I. differentiate the compositions that is used for the treatment of described experimenter;

Ii. modify described compositions by the lipotropy and the alkalescence that strengthen described compositions;

Iii. the experimenter who described modified compositions is needed treatment.

44. one kind is used for the treatment of experimenter's the method that needs can pass the compositions of blood brain barrier, comprises:

I. differentiate the compositions that is used for the treatment of described experimenter;

Ii. modify described compositions by the lipotropy, glycosylation and the alkalescence that strengthen described compositions;

Iii. the experimenter who described modified compositions is needed treatment.

45. one kind is used for the treatment of experimenter's the method that needs can pass the compositions of blood brain barrier, comprises:

I. differentiate the compositions that is used for the treatment of described experimenter;

Ii. modify described compositions by the lipotropy, glycosylation and the alkalescence that strengthen described compositions;

Iii. described modified compositions is inserted in the carrier;

Iv. the experimenter who described carrier is needed treatment.

46. a method for the treatment of the experimenter of the compositions that need be able to pass blood brain barrier comprises:

I. differentiate the compositions that is used for the treatment of described experimenter;

Ii. modify described compositions by the lipotropy and the alkalescence that strengthen described compositions;

Iii. described modified compositions is inserted in the carrier;

Iv. the experimenter who described carrier is needed treatment.

47. one kind prepares the method for compositions with enhanced blood-brain barrier permeability, comprise a kind of compositions with enhanced blood-brain barrier permeability of preparation, wherein said compositions comprises a kind ofly compares the peptide with enhanced lipophilic characteristic and enhanced alkalescence with this peptide of not transformed.

48. the method for claim 47, wherein said lipophilic characteristic can be strengthened by described peptide and hydrophobic part are puted together.

49. the method for claim 47, wherein said hydrophobic part is poly-aliphatic chain.

50. the method for claim 47, wherein said lipophilic characteristic can be strengthened by the halogenation that increases aromatic moieties.

51. the method for claim 47, wherein said alkalescence can be strengthened by same oligomer and the different oligomer of introducing the positive electricity amino acid residue, and described positive electricity amino acid residue includes but not limited to the ornithine of lysine, arginine, high-lysine, homoarginine, L-or D-isomer configuration; 2, the 3-diaminopropionic acid; 2,4-diamino-butanoic.

52. the method for claim 47, wherein said alkalescence can be by being strengthened with puting together based on the part of polyamines, described part based on polyamines is spermine, spermidine, polyamide-amide type dendrimer or polyamines toxin and derivant thereof for example.

53. the method for claim 47, wherein said peptide is compared with this peptide of not transformed, and the efficient of passing blood brain barrier can improve 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

54. the method for claim 47 comprises that also described peptide compares with this peptide of not transformed and also have enhanced glycosylation.

55. the method for claim 47, wherein said peptide contains sept.

56. the method for claim 55, wherein said sept is selected from: Gly, Ahx, Gly-Ahx or PEG-020c.

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