CN109718363A - Prevention, the peptide for alleviating or treating Alzheimer disease and its application - Google Patents

Abstract

This application provides the application of the peptide containing amino acid sequence YEKLLDTEI (SEQ ID NO:1) or its functional variant thereof or the pharmaceutical composition comprising the peptide in prevention, alleviation or treatment Alzheimer disease.

Description

Prevention, the peptide for alleviating or treating Alzheimer disease and its application

Technical field

The application relates generally to medical domain, specifically, this application provides prevention, alleviation or treatment alzheimer 's The peptide for disease of writing from memory and its application.

Background technique

Alzheimer disease (Alzheimer ' s disease, AD) is the more typical disease of person in middle and old age, is to be only second to heart and brain The disease for seriously threatening the elderly's life and health after vascular diseases, tumour.Clinical manifestation is progressive cognition dysfunction It is damaged with behavior.The pathogenesis of Alzheimer disease is related with mutated gene, internal and external environment and energetic supersession.

Under normal physiological condition, the level of Protein tau is more much lower than aixs cylinder in dendron.Under some diseases state, tau egg It is white middle exception to be enriched in dendron, the Alzheimer disease early stage especially mediated in A amyloid beta.Protein tau It needs to cooperate with Fyn and play a role, therefore in Δ tau74 and tau-/- mouse, the dendron of Fyn targets significant reduction.In Δ In tau74 mouse, this is because the binding site of Δ tau and endogenous tau competition and Fyn, plays the role of Reverse transcriptase. Protein tau -/- it is small in vivo in, there is similar phenomenon in the accumulation of Fyn, shows that postsynaptic Fyn targeting needs endogenous tau to mediate. Δ tau74 mouse and tau-/- mouse postsynaptic Fyn level reduce the Y1472 phosphorylation degree that can lead to Fyn- substrate NR2b It reduces.The Y1472 phosphorylation of NMDAR2B is the key condition for promoting the interaction of NMDAR and PSD-95.Fyn and Protein tau The NMDAR/PSD-95 compound formation of mediation is the precise mechanism in rats with stroke model, targetedly blocks NMDAR/ PSD-95 interaction prevents Excitotoxicity and reduces size of tumor.It is consistent with this, what A amyloid beta mediated AD early stage, the theory are equally applicable.To Δ tau74 and tau-/- mouse, after being treated using Tat-NR2B9c, NMDAR is situated between The synaptic activity and indifference led.

Exitotoxicity is gradually considered as that A amyloid beta causes one of mechanism of Alzheimer disease.Δ tau74 and Tau-/- mouse is compared with wild type, to excitatory toxicity and insensitive.Similarly, the primary mind of tau defect or expression Δ tau The toxicity of A amyloid beta induction can also be immunized through member.However, A amyloid beta level and patch are formed and APP23 The endogenous tau phosphorylation (Δ tau74 in APP23) of mouse shows another mechanism of protection compared with APP mouse.APP23 The Y1472 phosphorylation and postsynaptic Fyn that NR2b is found in mouse raise in APP23. Δ tau74 and APP23.tau-/- mouse It restores completely.APP23. Δ tau74 and APP.tau-/- mouse postsynaptic Fyn downward can show that dendron shape Fyn positioning also relates to And other tau mechanism, i.e. part Δ tau Reverse transcriptase Fyn positioning.In addition to participating in alzheimer ' caused by A amyloid beta Outside silent disease pathomechanism, there are epileptic attack premature deaths for Fyn transgenic mice.Neuronal cell is all referred in damaging pathways to wither It dies and inflammation caused by microglia, Neuron Apoptosis access Glutamic Acid receptor and postsynaptic densification albumen.In addition, APP Related mortality is substantially reduced on fyn-/- individual.APP23. Δ tau74 and the consistent performance of APP23.tau-/- mouse, says Bright routine Protein tau missing, effectively inhibits related defects caused by A amyloid beta.

NMDAR/PSD-95 compound in A amyloid beta toxic mechanism, plays a key effect in APP23 mouse, makees For the downstream molecules of Protein tau in Alzheimer disease pathologic, which may be related with tau mechanism and non-tau mechanism Connection.Applying A amyloid beta toxicity caused by TatNR2B9c, Protein tau and Fyn to young APP23 mouse can obtain Effectively inhibit.TatNR2B9c neuroprotection in vitro and in vivo relies primarily on the shape for inhibiting NMDAR/PSD-95 compound At equally capable of also improving the memory function and survival rate of APP23 mouse.It is worth noting that, the APP23 of Tat-NR2B9 treatment Mouse energy long-term surviving, this shows that short is enough to prevent A amyloid beta toxicity.

In treatment of alzheimer field, study of pathogenesis is constantly in progress, and treatment of alzheimer field is still It needs to meet the needs for the treatment of market according to new research and development therapeutic agent.

Summary of the invention

In a first aspect, this application provides become containing amino acid sequence YEKLLDTEI (SEQ ID NO:1) or its functionality The peptide of body or pharmaceutical composition comprising the peptide in preparation prevention, alleviate or the drug of the Alzheimer disease for the treatment of individual in Purposes.

Second aspect, this application provides prevention, alleviation or the methods for treating Alzheimer disease, which comprises To individual peptide of the application containing amino acid sequence YEKLLDTEI or its functional variant thereof in need or the medicine comprising the peptide Compositions.

The third aspect, this application provides become containing amino acid sequence YEKLLDTEI (SEQ ID NO:1) or its functionality The peptide of body or pharmaceutical composition comprising the peptide, are used for the Alzheimer disease for the treatment of, ameliorating or preventing individual.

In some embodiments of any of the above-described aspect, functional variant thereof is that the part LDTEI in YEKLLDTEI occurs The variant that one or more conservatives generate after replacing, it is preferable that the conservative replaces selected from the substitution between D and E, L, V The substitution between substitution and T and S between I.

In some embodiments of any of the above-described aspect, functional variant thereof is that the part LDTEI in YEKLLDTEI is replaced Be changed to the variant generated after following any sequences: LDTEL, LDTEV, LDTDI, LDTDL, LDTDV, LDSEI, LDSEL, LDSEV, LDSDI、LDSDL、LDSDV、LETEI、LETEL、LETEV、LETDI、LETDL、LETDV、VDTEI、VDTEL、VDTEV、 VDTDI、VDTDL、VDTDV、IDTEI、IDTEL、IDTEV、IDTDI、IDTDL、IDTDV、IETEI、IETEL、IETEV、 IETDI、IETDL、IETDV。

In some embodiments of any of the above-described aspect, peptide is comprising amino acid sequence YEKLLDTEI or its functionality Variant and be internalized by peptide chimeric peptide, the preferably described internalization peptide comprising amino acid sequence YGRKKRRQRRR (SEQ ID NO: 2)。

In some embodiments of any of the above-described aspect, chimeric peptide includes amino acid sequence YGRKKRRQRRRYEKLLDTEI(SEQ ID NO:3)。

In some embodiments of any of the above-described aspect, peptide, pharmaceutical composition or drug can improve Alzheimer Learning ability, memory capability (such as short term memory capacity) and/or the Spatial cognitive Abilities of sick diseased individuals.

In some embodiments of any of the above-described aspect, individual is mammal.

In some embodiments of any of the above-described aspect, individual is the mankind.

Detailed description of the invention

Fig. 1 shows the interaction of Pull-down experiment detection P5 and PDZ1/2 structural domain.M represents protein molecule Amount mark；Swimming lane 1 is His+PDZ1/2+P5；Swimming lane 2 is individual P5；Swimming lane 3 is His+P5；Swimming lane 4 is His+PDZ1/2. Elution band shown in swimming lane 1 includes both P5 and PDZ1/2, it was demonstrated that P5 can combine PDZ1/2 structural domain.

Fig. 2 show different therapeutic schemes to the test result of Alzheimer disease model rat escape latency, wherein " ★ " indicates p < 0.05.

Fig. 3 show different therapeutic schemes to the test result of Alzheimer disease model rat spanning platform number, In " ★ " indicate p < 0.05, " ★ ★ " indicate p < 0.01.

Detailed description of the Invention

Present inventor is to can reduce the neurological disorders at least partly mediated by NMDAR excititoxic The peptide of damage effect has made intensive studies, and develops a new class of peptide with the effect.It is not intended to by any theory Constraint, it is believed that this kind of peptide is at least partially by the interaction inhibited between NMDAR and 95 albumen of postsynaptic density (PSD-95) Come play a role (i.e. PSD-95 inhibitor).On this basis, present inventor is target spot to this using Alzheimer disease Class peptide is tested and has obtained ideal result.

The pathogenesis of Alzheimer disease is constantly explored in this field, and establishes a variety of theories, for example, refreshing The Crack cause of effect and amyloid protein through first inflammatory process in pathology and effect.But no matter amyloid protein is For the reason of Alzheimer disease pathologic starts still as a result, from amyloid protein to inflammatory reaction, centre will undergo neuron Apoptotic process.It is not intended to be bound by any theory, applicant thinks that Glu-induced Injury approach is wherein important channel, NMDAR- PSD95 is the important compound of the access, and the compound is inhibited to be formed, and can inhibit to a certain degree Neuron Apoptosis and be likely to become Influence the means of Alzheimer disease pathologic process.

Therefore, this application provides can be effective for prevention, alleviation and the drug and method for the treatment of Alzheimer disease Patients with Alzheimer disease symptom improves life quality and provides new selection.

Definition

Unless otherwise specified, term use herein has the normally understood meaning of those skilled in the art.

Single-letter or the trigram abbreviation used in the application for amino acid follows international usages.

Term " chimeric peptide " indicates tool there are two the peptide of component peptide not being bonded to each other naturally, and described two component peptides can be with It is bonded to each other as fusion protein or by chemical bond.

Term " PDZ structural domain " refers to the modular proteins matter structural domain of about 90 amino acid, it is characterized in that brain cynapse egg White PSD-95, drosophila (Drosophila) separate connection protein D iscs-Large (DLG) and connect albumen Z01 with epithelial tight (Z01) there is the sequence identity of significant (for example, at least 60%).PDZ structural domain is also referred to as the repetition of Discs-Large homology (" DHRs ") and GLGF are repeated.PDZ structural domain usually show retain core consensus sequence (Doyle, D.A., 1996, Cell 85: 1067-76).Illustratively the protein of the structural domain containing PDZ and PDZ domain sequence are in U. S. application No.10/714,537 It is open.

Term " nmda receptor " or " NMDAR " refer to the known film related protein with NMDA interaction.These receptors can To be people or inhuman (such as mouse, rat, rabbit and monkey etc.).

Term " specific binding " refers to the combination between two molecules (such as ligand and receptor), it is characterized in that even existing There are when many other different moleculars, the ability that a kind of molecule (ligand) and another special molecular (receptor) combine is being divided Show a kind of molecule to the ability of another molecule preferentially combined in the heterogeneous mixture of son.The specific binding of ligand and receptor Also be proved to as follows: when there is excessive unlabelled ligand, the combination reduction of ligand and receptor through detectable label (is combined Competitive assay).

Statistically significant refers to p value < 0.05, preferably < 0.01, most preferably < 0.001.

Term " functional variant thereof " refers to the variant for having same or similar biological function and property with parent.As Unrestricted example, " functional variant thereof " can replace acquisition by carrying out one or more conservatives in parent.

Term " internalization peptide " is alternatively referred to as cell-penetrating peptide, is widely used in pharmaceutical grade protein field, function be promote with The active peptide that it is combined is by cellular uptake and absorption.As a unrestricted example, being internalized by peptide can be Tat peptide, wherein One non-limiting example of Tat peptide is YGRKKRRQRRR (SEQ ID NO:2).

In a first aspect, this application provides become containing amino acid sequence YEKLLDTEI (SEQ ID NO:1) or its functionality The peptide of body or pharmaceutical composition comprising the peptide in preparation prevention, alleviate or the drug of the Alzheimer disease for the treatment of individual in Purposes.

Second aspect, this application provides prevention, alleviation or the methods for treating Alzheimer disease, which comprises To individual peptide of the application containing amino acid sequence YEKLLDTEI or its functional variant thereof in need or the medicine comprising the peptide Compositions.

The third aspect, this application provides become containing amino acid sequence YEKLLDTEI (SEQ ID NO:1) or its functionality The peptide of body or pharmaceutical composition comprising the peptide, are used for the Alzheimer disease for the treatment of, ameliorating or preventing individual.

The peptide is also referred to as " active peptide " in this application, and therapeutic activity portion is used as in the chimeric peptide of the application Point.

According to existing research, some active peptides for inhibiting the interaction between NMDAR and PSD-95 are based on NMDAR Structure.For example, NMDAR2B has GenBank ID4099612,20 amino acid of C-terminal are FNGSSNGHVYEKLSSLESDV and PL motif ESDV.Existing some active peptides have chosen the part ammonia of the C-terminal of NMDAR2B Base acid sequence, to generate the Reverse transcriptase to PSD-95 with NMDAR2B.Have research think the ESDV in above-mentioned peptide or LESDV section plays a significant role in inhibiting the interaction between NMDAR and PSD-95 albumen.Not by any theoretical beam In the case where tiing up, present inventor surprisingly it has been found that active peptide YEKLLDTEI disclosed herein (SEQ ID NO: 1) in, the C-terminal amino acid relative to above-mentioned NMDAR2B is formed, without containing two residues of SS after KL, while relative to PL motif increases the YEKL amino acid sequence in N-terminal direction, and the application confirms that such change can enhance active peptide and PDZ1/ The interaction of 2 structural domains.Simultaneously relative to YEKL motif, the LDTEI of C-terminal can be changed, it is contemplated that not influence activity The activity of peptide is likely to increase its activity.Therefore, in some embodiments, functional variant thereof provided by the present application is SEQ The variant generated after one or more conservatives replace occurs for the part LDTEI in ID NO:1.

In some embodiments, conservative replace substitution and T selected from substitution between D and E, between L, V and I and Substitution between S.

In more specific some embodiments, functional variant thereof is that the part LDTEI in SEQ ID NO:1 is replaced by The variant generated after following any sequences: LDTEL, LDTEV, LDTDI, LDTDL, LDTDV, LDSEI, LDSEL, LDSEV, LDSDI、LDSDL、LDSDV、LETEI、LETEL、LETEV、LETDI、LETDL、LETDV、VDTEI、VDTEL、VDTEV、 VDTDI、VDTDL、VDTDV、IDTEI、IDTEL、IDTEV、IDTDI、IDTDL、IDTDV、IETEI、IETEL、IETEV、 IETDI、IETDL、IETDV。

In some embodiments, functionality disclosed herein variant further includes having at least with above-mentioned peptide 60%, 70%, 75%, 80%, 85%, 90%, the 95%, amino acid sequence of even higher identity.It is known in the art, two " identity " between kind of albumen passes through the second hatching egg for replacing a kind of amino acid sequence of albumen and its conserved amino acid White sequence is compared to determine.Using well known to a person skilled in the art computerized algorithm and method determine two kinds of albumen it Between identity degree.Identity between two amino acid sequences preferably passes through to be determined using BLASTP algorithm.

In some embodiments, functionality disclosed herein variant include compared with above-mentioned peptide, have 1, 2, the substitution of the amino acid residue at 3,4,5 or more places, missing, addition and/or insertion are different from specific peptide disclosed above.

As described above, functional variant thereof can be replaced by one or more, be lacked, addition and/or insertion are different from State disclosed specific peptide.These variants can be naturally occurring or can be and be synthetically produced, for example, passing through modification one Or multiple above-mentioned peptide sequences disclosed herein and according to described herein with any one of multiple technologies well known in the art Assess its bioactivity.

In some embodiments, peptide is to become comprising amino acid sequence YEKLLDTEI (SEQ ID NO:1) or its functionality The chimeric peptide of body and internalization peptide, the internalization peptide can promote the chimeric peptide by cellular uptake.

It will be appreciated by those skilled in the art that the chimeric purpose of active peptide and internalization peptide, which is essentially consisted in, keeps active peptide more preferable Ground reaches action target spot, and therefore, the internalization peptide suitable for the application is not limited to particular types, wears film, interior as long as being able to achieve The purpose of change.It should also be appreciated by one skilled in the art that the action target spot due to active peptide is predominantly located at neuronal cell Inside, therefore the internalization peptide that can be specifically suitable for neuronal cell is preferred.In some embodiments, internalization peptide can Think Tat peptide.In some embodiments, the amino acid sequence of Tat peptide is YGRKKRRQRRR (SEQ ID NO:2).Some In embodiment, chimeric peptide includes amino acid sequence YGRKKRRQRRRYEKLLDTEI (SEQ ID NO:3).

It should be appreciated that internalization peptide can be keyed by amide as fusogenic peptide with active peptide, but can also pass through Other suitable modes are engaged, such as chemical bond engagement.The coupling of two kinds of components can be real by coupling agent or conjugation agent It is existing.A large amount of this kind of reagents are available commercial, and may refer to S.S.Wong, Chemistry of Protein Conjugation and Cross-Linking, CRC Press (1991).Some examples of cross-linking reagent include J- succinyl Imines -3- (2- pyridine two is thio) propionate (SPOP) or N, N'- (1,3- phenylene) bismaleimide；N, N '-ethylidene- Double-(iodoacetamide) or other this kind of reagents with 6 to 11 carbon methylene bridges (other sulfydryls are relatively special)；With 1,5- bis- Fluoro- 2,4- dinitrobenzene (it forms irreversible connection with amino and tyrosine-based).Other cross-linking reagents include P, P '-two Fluoro- m, m '-diphenylsulfone dinitro (it forms irreversible crosslinking with amino and phenolic group)；(its is right for caproic acid dimethyl ester for diethylamine Amino is special)；Phenol-Isosorbide-5-Nitrae-disulfonic acid chloride (it is mainly reacted with amino)；1, hexamethylene-diisocyanate or two different sulphur cyanogen Acid esters or the p- diisocyanate of phenylazo-(it is mainly reacted with amino)；(it is anti-from several different side chains for glutaraldehyde Answer) and double diazo benzidine (its mainly with tyrosine and histidine reaction).

In addition, previously described peptide can be derivatized (such as acetylation, phosphorylation and/or glycosylation) optionally to promote Into the affinity with inhibitor, inhibitor is promoted to cross over the ability that cell membrane is transported, or promotes stability.

The fusogenic peptide that the active peptide of the application can be synthesized by synthesis in solid state or recombination method and merged with internalization peptide.It can Peptidomimetic, the scientific literature and patent document example are synthesized using kinds of schemes described in scientific literature and patent document and method For example (eds.) John Wiley&Sons, Inc., the NY such as Organic Syntheses Collective Volumes, Gilman, al-Obeidi(1998)Mol.Biotechnol.9:205-223；Hruby (1997) Curr.Opin.Chem.Biol.1:114- 119；0stergaard(1997)Mol.Divers.3:17-27；0stresh(1996)Methods Enzymol.267:220- 234。

In some embodiments, active peptide or chimeric peptide disclosed herein can be applied in the form of pharmaceutical composition With.Pharmaceutical composition can by conventional mixing, dissolution, pelletize, ingot processed, grinding, emulsification, encapsulating, capture or freeze drying process Manufacture.

It is acceptable convenient for active peptide or chimeric peptide be processed into pharmaceutical preparations that one or more physiology can be used Carrier, diluent, excipient or auxiliary material, compounding pharmaceutical composition in a usual manner.It is appropriate to prepare applying dependent on selection Use approach.

In some embodiments, application can be parenteral, intravenous, oral, subcutaneous, intra-arterial, encephalic, it is intrathecal, Peritonaeum is interior, local, intranasal or intramuscular.It is preferred that intravenous application.

In some embodiments, it is preferably sterile and substantially isotonic for the pharmaceutical composition of parenteral administration. For injection, active peptide or chimeric peptide can be formulated into aqueous solution, preferably be formulated into the compatible buffer of physiology Such as (mitigate the discomfort at injection site) in Hank ' s solution, Ringer ' s solution or physiological saline or acetate buffer. Solution can contain preparaton such as suspending agent, stabilizer and/or dispersing agent.

Alternatively, active peptide or chimeric peptide can be for using preceding with suitable carrier (such as aseptic without heat source water) The powder type of building.

For across mucosal administration, the penetrating agent for being suitble to the barrier to be penetrated is used in formulation.The administration method can It is used to for compound to be delivered to nasal cavity or be used for sublingual administration.

It in some embodiments, can be by active peptide or chimeric peptide and pharmaceutical carrier one for oral administration It rises and is formulated as tablet, pill, pastille, capsule, liquid, gel, syrup, slurry, suspension etc., for being passed through by treated patient Mouth intake.For oral administration solid formulation such as powder, capsule and tablet, suitable excipient include filler for example Sugar, such as lactose, sucrose, mannitol and D-sorbite；Cellulose preparation such as cornstarch, wheaten starch, rice starch, potato Starch, gelatin, tragacanth, methylcellulose, carboxylic propyl methocel, sodium carboxymethylcellulose and/or povidone (PVP)；Granulation agent and adhesive.If desired, disintegrating agent can be added, such as polyvinylpyrrolidone, the agar of crosslinking, or Alginic acid or its salt, such as sodium alginate.Sugared package or enteric are carried out to solid dosage forms if desired, standard technique can be used Clothing package.For oral liquid such as suspension, elixir and solution, suitable carrier, excipient or diluent packet Include water, glycerol, oil, alcohol.Furthermore it is possible to add flavoring agent, preservative, colorant etc..

Other than previously described formulation, active peptide or chimeric peptide can also be configured to storage preparation.It can lead to It crosses implantation (such as subcutaneously or intramuscularly) or applies this kind of long-acting formulation by intramuscular injection.Thus, for example can be by compound It is prepared with suitable polymeric material or hydrophobic material (such as the emulsion being formulated as in acceptable oil) or ion exchange resin Together, or it is formulated as slightly molten derivative, such as is formulated as slightly molten salt.

Alternatively, other drugs delivery system can be used.Liposome and emulsion can be used to deliver chimeric peptide.Also it can be used Certain organic solvents such as dimethyl sulfoxide.Further, it is possible to use (such as solid containing therapeutic agent is poly- for the system of sustained release Close the semipermeability matrix of object) delivering compound.

According to its chemical property, sustained-release capsule releasable chimeric peptide several weeks are until more than 100 days.According to therapeutic reagent Chemical property and biological stability, can be used for protein stabilization other strategy.

In some embodiments, because of the side chain of active peptide disclosed herein or chimeric peptide containing electrification or end, So they can be used as free acid or alkali or are included in any of above formulation as pharmaceutical salt.Pharmaceutical salt is The basic bioactivity for retaining free alkali and by with the salt that is prepared and inorganic acid reaction.Drug salts are tended to dissociate than accordingly Alkali form is more soluble in water and other proton solvents.

In some embodiments, dosage range includes 0.001 to 20 μm of ol active peptides of every kg patient's weight or chimeric peptide, Optionally every 0.03 to 3 μm of ol active peptides of kg patient's weight or chimeric peptide.In certain methods, every kg patient's weight 0.1- is applied 20 μm of ol active peptides or chimeric peptide.In certain methods, 0.1-10 μm of ol active peptide of every kg patient's weight or chimeric peptide are applied, more It is preferred that applying every kg patient's weight about 0.3 μm of ol active peptide or chimeric peptide.In other cases, dosage range is every kg patient's body Weigh 0.005 to 0.5 μm of ol active peptide or chimeric peptide.It can be by compensating different surface area: mass ratio divided by 6.2, it will be every It is people that the dosage of kg weight is converted from rat.In gram, the suitable dose for the active peptide of people or chimeric peptide may include 0.01 To 100mg/kg patient's weight or more preferable 0.1 to 30mg/kg patient's weight or 1 to 10mg/kg (for example, 1,2,3,4,5,6, 7、8、9、10mg/kg)。

In some embodiments, the amount of the active peptide or chimeric peptide of application depends on treated subject, subject Weight, the seriousness of disease, method of application and the doctor to prescribe adjusting.It is when symptom can detect or even undetectable When repeatable treatment.Treatment can be provided separately or combine with other drugs offer.

In some embodiments, effective dosage is capable of providing and controls in the treatment of active peptide or chimeric peptide disclosed herein Benefit is treated without causing great toxicity.It can be measured in cell culture or experimental animal by standard pharmaceutical procedures chimeric The toxicity of peptide, such as pass through measurement LD50 (dosage for keeping 50% group lethal) or LD100 (dosage for keeping 100% group lethal) To realize.The dose ratio of poisonous effect and response to treatment is therapeutic index.The chimeric peptide of preferred display high therapeutic index is quasi- Peptide (see such as Fingl et al., 1975, in The Pharmacological Basis of Therapeutics, the 1st chapter, the 1st Page).

In some embodiments, pharmaceutical composition is the form of pre-lyophilization formulation or the form of lyophilized preparation, or freeze-drying Preparation combined with aqueous solution and obtain recovery preparation form.

It should be appreciated that be discussed in detail above only for making those skilled in the art more clearly understand present context, And it is not intended to limit in any way.Those skilled in the art can carry out various changes and change to the embodiment Change.

Embodiment

There is provided following embodiment is that only some embodiments of the application are illustrated, without any restrictions Purpose or property.

Embodiment 1: the screening of bioactive peptide molecule

According to reported result of study, choose Tat cell-penetrating peptide YGRKKRRQRRR (SEQ ID NO:2), and by its with not Amino acid with number is connected, and forms peptide library.By the peptide chimeric molecules in peptide library, respectively with vivoexpression and purify The interaction of PDZ1/2 structural domain carries out preliminary screening to polypeptide according to the power of interaction force.

Fixed molecule (ligand) is PDZ1/2 albumen, molecular weight :~20kD, concentration: 2mg/ml；The molecule of mobile phase (analyte): polypeptide to be screened, molecular weight :~2kD, concentration: 10mg/ml.Using 3000 instrument of Biacore, CM5 chip into Row is fixed.Electrophoretic buffer is PBS+0.005% polysorbas20.It is fixed using amino coupled method.The concentration of ligand is 10 μ g/ml.Fixed buffer is 10mM sodium acetate, pH 4.0.Fixed amount: 1400RU is fixed to flow cell 2.The flow velocity used is 10 μ l/ml, ligand sample introduction 1 minute.Use the 10mM Gly of PH 2.0+2.5 as regenerated liquid, with 30 l/ minutes flow velocitys of μ into Row regeneration.Sample injection time is 30s.

Dynamic analysis is carried out using following conditions: control channel: flow cell 1；Electrophoretic buffer is PBS；It uses Kinetic Analysis Wizard mode, concentration gradient 6.25n, 12.5n, 25n, 50n, 100n, 200n, 400nM；Into The sample time is 1 minute；Dissociation time is 2min；Flow velocity is 30 μ l/ minutes.

Data are fitted with 4.1 software of software BIAevaluation is fitted.Fitting model is 1:1 binding model. Dissociation constant KD value and active force are in inverse ratio.

By screening, obtains with PDZ1/2 structural domain with the chimeric peptide compared with strong interaction ability, be named as P5, sequence are as follows:

P5:YGRKKRRQRRRYEKLLDTEI(SEQ ID NO:3)

In order to which the direct similar chimeric peptide with reported research is compared, control chimeric peptide NA-1, sequence are introduced It arranges as follows:

NA-1:YGRKKRRQRRRKLSSIESDV

In addition, by comparing the architectural difference of P5 and NA-1, the N-terminal in addition introduced in the active peptide of chimeric peptide NA-1 adds Enter the chimeric peptide YE-NA-1 of two residues of YE, sequence is as follows:

YE-NA-1:YGRKKRRQRRRYEKLSSIESDV

Chimeric peptide NA-1, YE-NA-1 and P5 are carried out to the survey described above with the interaction of PDZ1/2 structural domain simultaneously Examination, as a result shown in table 1 as follows:

1. 3 kinds of chimeric peptides of table and PDZ1/2 structural domain interaction force detect

Chimeric peptide	NA-1	YE-NA-1	P5
				KD(M)	7.53E-08	5.44E-08	2.99E-08

As shown in table 1, compared to control chimeric peptide NA-1, chimeric peptide YE-NA-1 and P5 and PDZ1/2 structural domain phase interaction It is firmly stronger, and the interaction property of P5 is more preferably.Therefore, according to the supposition of inventor, additional two amino of YE of the N-terminal of active peptide Sour residue has certain humidification to the interaction of polypeptide and PDZ1/2 structural domain.In addition, carboxylic of the P5 relative to YE-NA-1 Cardinal extremity reduces two weaker serines of hydrophobicity (SS), and according to the supposition of inventor, therefore this may further increase more The interaction of peptide and PDZ1/2 structural domain.

Chimeric peptide P5 is further tested in experiment below, and is made NA-1 and YE-NA-1 in part Experiment For control.

The interaction of embodiment 2:Pull-down experiment detection P5 and PDZ1/2 structural domain

To prove that P5 can interact with PDZ1/2 structural domain, Pull-down experiment is carried out.

Pillar is balanced into 5min with the MCAC-0 buffer of the His pearl of 100 μ l and 1ml.It is shaken at 4 DEG C.By mixture At 4 DEG C, with 5000g centrifugation 1 minute, supernatant is abandoned.1mg PDZ1/2 albumen is added into mixture, and extremely with buffer polishing 1ml.At 4 DEG C, mixture rotation is combined 1 hour.By the mixture at 4 DEG C, with 5000g centrifugation 1 minute, in abandoning Clearly.With MCAC-0 buffer solution for cleaning 3 times of 1ml, 5 minutes every time (at 4 DEG C, concussion washing).1mg P5 is added into mixture Albumen, and with buffer polishing to 1ml.At 4 DEG C, mixture rotation is combined 2 hours.By the mixture at 4 DEG C, with 5000g is centrifuged 1 minute, abandons supernatant.Clean 3 times, 5 minutes every time (at 4 DEG C, concussion washing) with 1ml lysate.Clean it After 20 μ l MCAC-300 are added.Centrifugation takes eluent to carry out SDS-PAGE detection.Experimental result is shown in Fig. 1.

If Fig. 1 is confirmed, simultaneously comprising both P5 and PDZ1/2 structural domains in the elution band of chimeric peptide P5, it is confirmed that Chimeric peptide P5 can combine PDZ1/2 structural domain.

Embodiment 3: acute toxicity test

Acute toxicity test is carried out with rat.The result shows that: under the dosage of 200mg/kg weight, P5 to rat without Lethal effect and other apparent toxic side effects.

Effect of the embodiment 4:P5 peptide to treatment Alzheimer disease

I. instrument

Instrument used in the present embodiment is as follows:

1056189 double end cold light sources (Beijing Zhong Shidichuan development in science and technology Co., Ltd).

TJ-4A micro-injection pump (Beijing Zhong Shidichuan development in science and technology Co., Ltd)

ZS-001morris water maze video analytic system (Beijing Zhong Shidichuan development in science and technology Co., Ltd)

ZS-GSZ sphenotresia instrument (Beijing Zhong Shidichuan development in science and technology Co., Ltd)

II. material

Material used in the present embodiment is as follows:

Physiological saline (Shijiazhuang Siyao Co., Ltd, national drug standard H13023200)；

Polypeptide (P5, NA-1 are prepared by Hangzhou Zhong Tai Co., Ltd)；

Chloraldurate (Sinopharm Chemical Reagent Co., Ltd., lot number 20150303)；

Gentamicin (Huazhong Pharmaceutical Co., Ltd., national drug standard H42021503)；

A amyloid beta (article No. A1075 is purchased from sigma (China))；

Dental cement (Beijing Zhong Shidichuan development in science and technology Co., Ltd)；

Memantine (Beijing Suo Laibao Biotechnology Co., Ltd).

III. experimental animal

The experimental animal that the present embodiment uses is 240g~280g SD rat, purchased from change Fukang at Co., Ltd.

IV. method and steps

1. rat screening and grouping

80 240~280 grams of male SD rats are selected as experimental mouse, are divided into 7 groups, every group 9 to 11,7 groups are respectively as follows: Normal group, model group (AD group), tri- dosage groups of P5 (tail vein injection 1mg/kg, 3mg/kg, 10mg/kg), NA-1 (tail vein Inject 3mg/kg, peptide medicine control), Memantine group (stomach-filling 2mg/kg, chemical drugs control).

2. modeling

1) 10% chloraldurate solution of SD rats by intraperitoneal injection is anaesthetized, dosage 4.5ml/kg.

2) rat head is fixed on stereotaxic apparatus, the operative site tincture of iodine+cotton ball soaked in alcohol routine disinfection, preserved skin.

3) elbow scissors cuts off epidermis, and blunt separates skin, subcutaneous tissue and periosteum, and exposure skull finds bregma position.

4) 3mm after label bregma, the left and right position 2mm one position of each label is polished with ZS-GSZ sphenotresia instrument and is marked Locate skull to sufficiently thin, at totally two, tries not to puncture, pointed out using 10 μ l micro syringes, then proceed to downward inserting needle 3.5 μm。

5) A amyloid beta is injected, 2.5 μ l/5min of injection parameters, let the acupuncture needle remain at a certain point after injection 5 minutes, the then slow withdraw of the needle, A β Amyloid protein is 1mg/ml.

6) at dental cement closing sphenotresia ,+4.0 operation suture thread of sword-shaped needle is sewed up a wound, and leg muscle injection celebrating is big Mycin 1mg/kg, commercialization stoste dilute three times.

7) with the operation same day for first day, development successive administration processing in the 10th day.

3. administration and test

It is tested in this research using Morris water maze (Morris water maze).The test is to force experimental animal The experiment for being hidden in underwater platform is found in swimming, study, and it is (empty to spatial position sense and sense of direction to be mainly used for test experiments animal Between position) ability of learning and memory, be widely used in Alzheimer disease drug evaluation research in.

Every group after modeling 10 days, starts successive administration 14 days, carries out water maze survey immediately once a day, after administration Examination, test in continuous 4 days are appeared on the stage incubation period, and entering water quadrant is to remove within the 4213,1234,2413,2143, the 5th day platform development to wear platform Experiment, entering water quadrant is 4321.

4. incubation period tests

Every rat of different grouping, which is tested every time from the same position, enters water, enters in water quadrant such as " 3. administrations and test " It is described.When rat enters water, operator mentions rat tail, and mouse head slowly into the water towards wall, find platform after entering water and stop by rat It stays 2 seconds, experiment stops, and the spent time is incubation period, did not found platform yet more than 120 seconds and is also denoted as 120 seconds.Regular replenishment Water, keeps platform apart from water surface 2cm, tests every time sart point in time sart point in time 1 minute away from test next time.

5. wearing platform experiment

After rat is according to the continuous 4 days water maze incubation periods test of quadrant shown in " 3. administrations and test ", start to remove water Platform in labyrinth, record rat enter the motion profile after water, intersect once to be denoted as with position of platform and once wear platform, record every Rat wears platform total degree in 120 seconds, enter water quadrant 4321.

V. statistical analysis

Every rat enters water 4 times in 4 quadrants daily, tests within continuous 4 days, is finally averaged obtain 16 numbers Value, for the final incubation period of this rat, the 5th day every rat enters water 4 times in 4 quadrants, only tests one day, will finally obtain 4 numbers be averaged, wear platform number for this rat.T-test function (mantissa 2, the type carried using EXCEL Incubation period and platform number is worn 3) to be respectively compared model group with appearing on the stage of normally organizing.P < 0.05 thinks there is statistical difference, p < Think there is significant statistical difference for 0.01.If normal group and model group (AD group) are in incubation period and wear platform number and have statistics Difference is learned, assert modeling success.

It it is respectively compared model group and each administration is again grouped and appear on the stage incubation period and wear platform number.Think have statistics poor in p < 0.05 Different, p < 0.01 item thinks there is significant statistical difference.If processing group and model group (AD group) are in incubation period or wear platform number and deposit In statistical difference, assert that processing group has therapeutic effect.

VI. experimental result

1. effect of the different dosing group to rat model escape latency

The escape latency statistical result of each group rat is referring to table 1 and Fig. 2.As the result is shown: p between normal group and AD group < 0.05 (unmarked in figure) illustrates modeling success.Three dosage groups (1mg/kg, 3mg/kg, 10mg/kg) of P5 and NA-1 group with AD group compares, p < 0.05, illustrates to present effect in this item test, and this four groups escape latency substantially with normal group Quite.Chemical drugs compare Memantine group and do not show effect in this item test.

2. the effect that each processing group tests rat spanning platform

The spanning platform number of each group rat is counted referring to table 2 and Fig. 3.As the result is shown: p between normal group and AD group < 0.05 (unmarked in figure) illustrates modeling success.Two dosage groups (3mg/kg, 10mg/kg) of P5 compared with AD group, p < 0.05, illustrate to generate therapeutic efficiency in this item test, and this two groups spanning platform number is substantially suitable with normal group.Change Medicine control Memantine group is learned compared with AD group, p < 0.01 illustrates also to generate therapeutic efficiency.Two effective dose group (3mg/ of P5 Kg and 10mg/kg) and Memantine group between no difference of science of statistics.Peptide compares NA-1 group and does not show effect in this item test.

VII. it discusses

Two dosage groups (3mg/kg and 10mg/kg) of P5 are in incubation period and wear platform number two tests and have system with AD group Meter learns difference (p < 0.05), and actual result is suitable with normal group, shows therapeutic efficiency.But it is similarly the peptide of 3mg/kg NA-1 group is compareed, effect is only shown in incubation period test, and does not have effect in wearing the test of platform number.Memantine is The drug of commercially available a treatment Alzheimer disease, only shows effect on wearing the test of platform number, and surveys in incubation period There is no effect in examination.Cumulated volume research as a result, the exemplary peptides P5 of the application have in treatment Alzheimer disease it is good Application prospect.

All publications and patents cited in this specification references cited therein as reference, such as each publication or Patent is clearly indicated respectively to be incorporated herein by reference.It, can without departing from spirit and scope disclosed in the present application Each embodiment disclosed in the present application is variously changed and is replaced with equivalent.Unless being otherwise noted in context, otherwise Any feature, step or the embodiment of the embodiment of the disclosure can be with any other feature, step or embodiments It is applied in combination.

Sequence table

<110>Bai Xiousi (Beijing) Bioisystech Co., Ltd

<120>prevention, the peptide of alleviation or treatment Alzheimer disease and its application

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<213>artificial sequence (Artificial Sequence)

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Claims (9)

1. the peptide containing amino acid sequence YEKLLDTEI (SEQ ID NO:1) or its functional variant thereof or the medicine comprising the peptide Purposes of the compositions in preparation prevention, the drug alleviated or treat individual Alzheimer disease.

2. prevention, the method alleviated or treat Alzheimer disease, which comprises contain ammonia to individual application in need The peptide of base acid sequence YEKLLDTEI or its functional variant thereof or pharmaceutical composition comprising the peptide.

3. the peptide containing amino acid sequence YEKLLDTEI (SEQ ID NO:1) or its functional variant thereof or the medicine comprising the peptide Compositions are used for the Alzheimer disease for the treatment of, ameliorating or preventing individual.

4. purposes as described in claim 1 or method as claimed in claim 2 or pharmaceutical composition as claimed in claim 3, Wherein the functional variant thereof is the change that the part LDTEI in YEKLLDTEI occurs to generate after one or more conservatives replace Body, it is preferable that the conservative replaces selected from the substitution between D and E, taking between the substitution and T and S between L, V and I Generation.

5. purposes as claimed in claim 4 or method or pharmaceutical composition, wherein the functional variant thereof is in YEKLLDTEI The part LDTEI be replaced by the variant generated after following any sequences: LDTEL, LDTEV, LDTDI, LDTDL, LDTDV, LDSEI、LDSEL、LDSEV、LDSDI、LDSDL、LDSDV、LETEI、LETEL、LETEV、LETDI、LETDL、LETDV、 VDTEI、VDTEL、VDTEV、VDTDI、VDTDL、VDTDV、IDTEI、IDTEL、IDTEV、IDTDI、IDTDL、IDTDV、 IETEI、IETEL、IETEV、IETDI、IETDL、IETDV。

6. such as the described in any item purposes of preceding claims or method or pharmaceutical composition, wherein the peptide is to include amino acid Sequence YEKLLDTEI or its functional variant thereof and the chimeric peptide for being internalized by peptide, the preferably described internalization peptide include amino acid sequence YGRKKRRQRRR(SEQ ID NO:2)。

7. purposes as claimed in claim 6 or method or pharmaceutical composition, wherein the chimeric peptide includes amino acid sequence YGRKKRRQRRRYEKLLDTEI(SEQ ID NO:3)。

8. such as the described in any item purposes of preceding claims or method or pharmaceutical composition, wherein the peptide, pharmaceutical composition Or drug can improve the learning ability of Alzheimer disease diseased individuals, memory capability (such as short term memory capacity) and/or Spatial cognitive Abilities.

9. such as the described in any item purposes of preceding claims or method or pharmaceutical composition, wherein the individual is dynamic for lactation Object, the preferably mankind.