CN104292308B - Tumour antigen cyclic peptide - Google Patents

Abstract

The present invention relates to a kind of tumour antigen cyclic peptide, it is therefore intended that provides a kind of cyclic peptide and its derivative, can induce immune response, attacks tumour cell.Formula (I) polypeptide of the present invention can be used alone or be used with pharmaceutical compositions.Application method can be oral or parenteral approach.Oral administration preparation can be tablet, capsule or granule etc..Non-intestinal drug delivery agent can be injection, spray, patch etc..The present composition may be made as sustained release preparation, such as microballoon.Optimizing injection.

Description

Tumour antigen cyclic peptide

Technical field

The present invention relates to cyclic peptide compounds or its salt, it is to including prostate cancer, lymph cancer, particularly lung cancer, breast cancer It is inhibited.

Its preparation method, the pharmaceutical composition comprising the cyclic peptide compounds or its salt and treatment human prostate cancer, The method of lymph cancer, particularly lung cancer, breast cancer.

Background technology

HER2/neu is the member of Epidermal Growth Factor Receptor Family and encodes 185kd participations regulation cell growth and numerous The tyrosine kinase receptor grown.HER/neu overexpression and/or amplification are found that in the tumour cell of many types.

By clinical research, discovery can target the epitope on HER2/neu tumor associated antigen, carry out cellular immunity The vaccine inoculation of response and effective immunization therapy.Because the immunocyte of body is not to correspond in various cause of diseases The overall molecule of body or native antigen, but for the epitope of various antigen molecule, proteantigen passes through its table Embody its immunologic opsonin in position.Immunologic intervention means based on antigen molecule have proceeded to epitope levels.As research the most Sufficient HER2/neu proto-oncogenes family, be found that thereon it is numerous can induce the epitope sequences of immune response, wherein Peptide sequence Lys-Ile-Phe-Gly-Ser-Leu-Ala-Phe-Leu (also known as E75) in its 369-377 position can be stimulated carefully Cytotoxic T Lymphocytes (CTL) destroy cancer cell, the vaccine as prevention and treatment tumour, are particularly suitable for use in breast cancer.But It is that E75 belongs to linear peptides, stability not enough, easily by proteasome degradation in blood plasma, can not very effectively be stimulated and exempted from vivo Epidemic disease system induction induces immune response.It is therefore desirable to peptide is transformed.

The content of the invention

In order to solve the above technical problems, it is an object of the invention to provide a kind of cyclic peptide and its derivative, can induce and exempt from Epidemic disease response, attacks tumour cell.

Annular closed or cyclisation can reduce the degradation rate of internal peptide, so as to substantially improve their pharmacokinetics Energy.

The technical scheme is that：

A kind of tumour antigen cyclic peptide, is represented by below general formula (I)：

Wherein X is following formula (Xa), (Xb) or (Xc)；

AA can be D types or the natural or alpha-non-natural amino acid of L-type.

N is homologue, preferential 1-3, in same molecule, and n can be different.

In the present invention, specifically,

AA is leucine, lysine, histidine, tryptophan, aspartic acid or arginine (other with diamine).

Wherein, preferred amino acid is leucine and lysine.

According to the preferred embodiments of the invention, cyclic peptide of the present invention is selected from following peptide or its pharmaceutical salts：

The present invention relates to include at least one formula (I) polypeptide or its pharmaceutical salts and the medicine group of pharmaceutical acceptable carrier or excipient Compound.

The invention further relates to formula (I) polypeptide or its stereoisomer or its pharmaceutical salts prepare treatment human prostate cancer, Purposes in lymph cancer, particularly lung cancer, breast cancer disease medicine.

The preparation of linear peptides of the present invention uses solid phase or liquid-phase synthesis process, the wherein preferred Fmoc protections plan of solid-phase synthesis Slightly, according to the amino acid sequence of prepared compound, from C-terminal to N-terminal, by standard method synthesizing linear peptide.

The preparation of cyclic peptide of the present invention using oxidizing process formation disulfide bond prepare, can using iodine oxidation method, air oxidation process or Other theoretical visible oxidants and operation.

Term used herein " officinal salt " includes：Hydrochloride, hydrobromate, sulfate, phosphate, nitrate, with And acetate, oxalates, tartrate etc..

According to the present invention, pharmaceutical composition of the invention can be prepared by this area conventional method, for example by formula (I) polypeptide with Pharmaceutical carrier or excipient mixing.Pharmaceutical carrier or excipient used are usually the conventional pharmaceutical carrier of pharmaceutical field in the present invention Or excipient.Here " conventional drug excipient " includes any or all solvents, and decentralized medium is coated, antiseptic or anti- Epiphyte pharmaceutical, isotonic and sustained release dosage, and similar physiology compatibility agent, to be adapted to intravenous injection, intramuscular injection, subcutaneous note Penetrate, or other administering modes, such as it is administered orally.According to the mode of administration, reactive compound can be coated to protect compound to exempt from Influenceed and inactivated by sour or other natural conditions.On the example of carrier, such as salt is water base and various aqueous buffer solutions, ethanol or Other polyalcohols, liposome, PLA, vinyl acetate, polyanhydride, polyglycolic acid, collagen, poe etc..

Formula (I) polypeptide of the present invention can be used alone or be used with pharmaceutical compositions.Application method can be oral or Parenteral route.Oral administration preparation can be tablet, capsule or granule etc..Non-intestinal drug delivery agent can for injection, Spray, patch etc..The present composition may be made as sustained release preparation, such as microballoon.Optimizing injection.

Brief description of the drawings

Fig. 1 is that cyclic peptide 1,2,3 of the present invention can effectively discharge the schematic diagram one of IFN-γ；

Fig. 2 is that cyclic peptide 1,2,3 of the present invention can effectively discharge the schematic diagram two of IFN-γ；

Fig. 3 is the specific CTL of epitope inducing peptide of the present invention to tumor cell specific lethal effect schematic diagram；

Fig. 4 is the elimination half-life period schematic diagram of linear peptides of the present invention and cyclic peptide 1,2,3,4.

Embodiment

With reference to embodiment, the embodiment to the present invention is described in further detail.Following examples are used for Illustrate the present invention, but be not limited to the scope of the present invention.

Embodiment 1

The synthesis of S1 linear peptides

S11 takes Rink Amide resins 1mmol, and (s=0.6mmol NH2/g, 100 mesh, the degree of cross linking is 1.0%), to be put into many In peptide symthesis reactor, deprotected with DMF swelling 30min, plus 20% piperidines (PIP/DMF), washing.

Cys (Trt), the HOBT that S12 protects Fmoc are completely dissolved with DMF, are added DIC and are well mixed, are added to reaction Reacted in device.Whole reaction stirring nitrogen charging, carries out 0.5-2h at room temperature.After Kaiser detections coupling reaction is complete, DMF is washed Dereaction solvent.Protected amino acid, HOBT, DIC molar ratio are 1:1:1.Resin and protected amino acid feed intake mole Than for 1:3.

S13 is sequentially ingressed into Fmoc-Cys (Trt), Fmoc-Leu, Fmoc-Phe, Fmoc-Ala, Fmoc-Leu, Fmoc-Ser (tBu)、Fmoc-Gly、Fmoc-Phe、Fmoc-Ile、Fmoc-Lys(Boc)、Fmoc-Cys(Trt)。

S14 drains peptide resin after completion of the reaction, weighs and about obtains 5g.Peptide resin is put in reactor, by 1g peptide resins: The ratio of 10ml lysates adds lysate (lysate：TFA:EDT:H2O:TIS=95:2:2:1), react at room temperature 2 hours, sand Core is filtered, and filtrate is collected, by 1:20 (v/v) ratio is slowly added into ice ether, precipitates 30min, and precipitation is collected in filtering, this Confirmed for linear peptides 1 and by mass spectrum：

Cys-Lys-Ile-Phe-Gly-Ser-Leu-Ala-Phe-Leu-Cys

Embodiment 2

The cyclisation of S2 peptides

S21 water dissolves linear peptides, makes linear peptide concentration to 0.5mg/ml or so, and stirring is lower to be added dropwise I2 liquid to having aoxidized Entirely, this produces cyclisation peptide crude product solution.

S22 carries out preliminary purification with low pressure purification system to cyclic peptide crude product.According to the content and volume of the thick peptide of each batch, press 1g peptides/50g resin ratio loadings, gradient elution collects purpose peak, will collect solution cross 0.22 μm of organic phase filter membrane, rotate it is dense Contracting, low temperature place standby.

S23 is refined with preparation solution relatively low pressure refined solution.Condition is as follows：

Mobile phase A：H2O+0.1%TFA；Mobile phase B：ACN+0.1%TFA

Flow velocity：50ml/min；Detection wavelength：214nm

Gradient：0~8min：5%B；

8~33min：22%B~30%B

33~35min：30%B~50%B

35~43min：50%B

Fractional Collections component, after detection is qualified, revolving, low-temperature dark is placed, standby.

S24 refined liquids slough TFA or turn salt, freeze, and obtain required cyclic peptide, MS identification products.

S25 presses above-mentioned standard program, obtains cyclic peptide 1：

S26 is on the basis of S13, and last peptide reaction is changed to introduce MPR groups after Fmoc-Lys (Boc).Will 300ml DCM are placed in ice bath in round-bottomed flask, and stirring adds Mpr 150ml reaction 5min, adds DIC 200ml stirring reactions Reaction solution, is then added in the reactor of the complete protected amino acid of above-mentioned company, stirring reaction by 25min, until ninhydrin detection is anti- Should be complete, stop reaction, washing is drained, and completes follow-up cracking, precipitation, cyclisation, purge process.Obtain cyclic peptide 2：

S27 is on the basis of S26, and C-terminal amino acid is Lys, connects Mpr, and synthesizing linear peptide simultaneously prepares cyclic peptide 3：

S28 is not by head and the tail disulfide bond cyclization, and head and the tail Lys and Leu directly by peptide bond cyclization, synthesizes control peptide (cyclic peptide 4):

Embodiment 3

S3CTL activation experiments

S31 utilizes enzyme linked immunological to confirm activation of the tumor antigen peptide cyclic peptide of synthesis in tumor cell line response Spotting method measures the generation of IFN-γ, the reading activated as cytotoxic T lymphocyte (CTL).

The preparation of S32 target cells

S33MCF-7 breast cancer and SBC-5 ED-SCLCs, purchased from ATCC.

The preparation of S34 effector cell

S35 obtains 1 using the negative separating kits (Dynal) of commercially available CD4 and CD8：The CD4+ and CD8+T of 1 mixing are thin Born of the same parents.The linear peptides and cyclic peptide 1,2,3,4 that Examples 1 and 2 are obtained be separately added into cell mixing cultivate 5 days after, obtain this hair Bright effector cell.

S36 ELISpots test (ELISPOT)

S37 takes the anti-IFN-γ capture antibody of 100 μ l to be diluted in the sterile PBS of 10ml, by 100 μ l capture antibody diluted etc. Part is added in each hole of PVDF plates.4 DEG C overnight.Then with lavation buffer solution washing flat board to remove capture antibody.Add per hole Enter sterile PBSs of the 100 μ l containing 2% skim milk with blocking of plates, by flat board in incubation at room temperature 2 hours, incline de- on flat board Fat milk.Then substances are added in ELISPOT flat boards in the following order：

50 106 cell/ml of μ l target cell (about 50,000 target cell/holes of meter).

50 μ l effector cells (meter about 5, the CD4/8+ cells/wells of the importing small peptide of 000 mixing).

S38 plates incubated overnights.Flat board was washed three times with lavation buffer solution in second day, it is unnecessary to remove to be patted with paper handkerchief Lavation buffer solution.Then 100 μ l primary detection antibody (people's IFN-γ ELISPOT PVDF enzymatic reagents are added into each hole Box, Diaclone companies production, takes the 100 μ l solution to be diluted in 10ml PBS and 1% bovine serum albumin(BSA)), flat board room temperature After being incubated 2 hours, washed with lavation buffer solution three times, patted to remove unnecessary lavation buffer solution with paper handkerchief.Add to each hole Enter the Streptavidin alkaline phosphatase of 100 μ l dilutions, then flat board is incubated at room temperature 1 hour, three are washed with lavation buffer solution It is secondary, patted to remove unnecessary lavation buffer solution with paper handkerchief.Then 100 μ l BCIP/NBT solution are added to each hole.Reacted Flat board is covered with paillon foil in journey, 5-15 minutes are stood.Inspect periodically flat board spot during this period, determine terminating reaction it is optimal when Between.Flat board is washed with terminating reaction in the tank for fill with running water, flat board is placed in after 50 DEG C of dryings 1 hour with immune spot Point plate reading machine counts the spot generated on film, obtains the linear peptides and cyclic peptide of the present invention.

As a result such as Fig. 1,2, cyclic peptide 1,2,3 can effectively discharge IFN-γ, possess immune response inductivity.

Embodiment 4

Detection of the specific CTL of S4 epitope inducing peptides to tumor cell specific lethal effect.

S41 is grouped：Effector cell is divided into 5 big group of samples (cyclic peptide 1,2,3,4, linear peptides 1), is lured respectively with each epitope peptide The specific CTL led is effector cell.Target cell：MCF-7 breast cancer and SBC-5 ED-SCLCs, 293 cells are thin as control Born of the same parents are, purchased from ATCC.

It is prepared by S42 effector cell：The specific CTL of TCL epitope inducing peptides is prepared by preceding method, with containing 5% tire ox blood Clear RMPI-1640 culture mediums are adjusted to debita spissitudo, are used as effector cell.

S43LDH is detected：Use Cytotox 96Non-Radioactive Cytotoxicicy Assay (cytotoxicities Detection kit, Promega companies) LDH experiment detection cytotoxic activities are carried out, key step is as follows.

S44 detection plates are set up：

Experimental group is using MCF-7 and SBC-5 as target cell (optimal target cell number is 5*103/ hole), by different effect target ratios 10:1、20:1、40:1 adds above-mentioned effector cell CTL, and various concentrations of cells are inoculated in 96 well culture plates to end with 50 μ l/ holes The μ l of volume 100.The spontaneous release group of effector cell：Each group effector cell contains 5% hyclone with 50 μ l/ orifice plates, 50 μ l of supplement RPMI-1640 culture mediums are to the μ l of final volume 100.The spontaneous release group of target cell：Each group target cell adds 96 orifice plates with the holes of 50 μ 1/, RPMI-1640 culture mediums of the 50 μ l containing 5% hyclone is supplemented to the μ l of final volume 100.Target cell maximum release group, same to target cell Spontaneous release group.Ground control group：Add the μ l of RPMI-160 culture mediums 100 containing 5% hyclone；Volume correction control group： Add the μ l of RPMI-1640 culture mediums 100 containing 5% hyclone.

S45 cells are cracked and harvest supernatant：After plating cells, 250g centrifugation 4min, 37 DEG C, 5%CO2, saturated humidity bar 4h is incubated under part.Lysate (10X) is added respectively at the maximum release group of target cell and volume correction control group, 10 μ l/ holes, After 45min, 250g centrifugation 4min harvest supernatant.

S46LDH is detected：Supernatant is shifted to another 96 orifice plate, 50 μ l/ holes；The Substrate cocktail of rapid addition dilution, 50 μ L/ holes, room temperature lucifuge reaction 30min；Add the reaction of terminate liquid color development stopping, 50 μ l/ holes；Take out in bubble in hole, 1h in enzyme mark Instrument detection 490nm light absorption values OD490.

S47 calculates cell killing rate

Killing rate (%)=[(the spontaneous spontaneous release group 0D of release group 0D values-target cell of experimental group 0D values-effector cell Value)/(the spontaneous release group 0D of target cell maximum release group 0D- target cells)] X100%

As a result：As shown in figure 3, the MCF-7 and SBC-5 that the CTL that cyclic peptide 1,2,3 is activated expresses the positive for Her2/neu are thin Born of the same parents are respectively provided with specific cytotoxicity, and express Her2/neu 293 negative cells then without obvious lethal effect, while ring Peptide 1,2,3 is substantially better than SBC-5 for the lethal effect of MCF-7 cells.

Based on above-mentioned experimental result, it is known that：Compared with linear peptides, cyclic peptide 1,2,3 more can the obvious specific CTL that must be induced Secretion of gamma-IFN, and certain antigentic specificity lethal effect is shown to MCF-7 cells and SBC-5 cells；And to MCF-7 The lethal effect of cell is substantially better than SBC-5 cells, shows that the tumor vaccine is more suitable for breast cancer.

Embodiment 5

S5 plasma elimination half lifes are determined.

In order to confirm that the tumour antigen cyclic peptide of synthesis has longer half-life period, embodiment 1 is detected using LC/MS/MS methods With the elimination half-life data of 2 linear peptides and cyclic peptide 1,2,3,4 obtained on the diagram.

LC/MS/MS development of methodology, Finnigan TSQ Discovery Max LC-MS instrument, is equipped with Surveyor HPLC system, detection is limited to 1-1000ng/mL, and the rate of recovery is more than 70%.

S51 takes new zealand rabbit, linear peptides and cyclic peptide that intravenous injection 0.5mg/kg Examples 1 and 2 are obtained.Respectively 5, 10th, 15,30,60,120,180,240,300 minutes difference blood sampling 1.0mL are in anticoagulant tube, and 10000rpm centrifugations separate bleeding Slurry, -20 DEG C of freezen protectives are until determine.

S52 determines the blood concentration of linear peptides and cyclic peptide using LC/MS/MS methods.Take blood plasma (blank plasma, plasma containing drug Or blood plasma after administration) 100 μ l, add the acetonitriles of 2000ng/ml containing IS：Methanol (70:30) the μ l of mixed solvent 500, vibration, 12000rpm centrifuges 10min, and supernatant is transferred in test tube, and less than 40 DEG C N2 dryings, residue adds 100 μ l MP to redissolve, 12000rpm centrifuges 5min, the μ l of supernatant sample introduction 20.Carry out LC/MS/MS analyses.Calculated with 3p97 pharmacokinetics calculation procedure Go out main pharmacokinetic parameter.

Measurement result shows that the linear peptides that embodiment 1 is obtained, elimination half-life period is 44 ± 8min or so, and embodiment 1 is obtained Cyclic peptide 1,2,3,4, it is respectively 123 ± 15min, 113 ± 18min, 105 ± 10min, 89 ± 9min or so to eliminate half-life period, Cyclic peptide 1,2,3 has clear improvement than the E75 rats elimination half-life period of document report less than 1h.The enclosed construction of cyclic peptide formation, The effect of protease in blood plasma can be preferably resisted, active compound form can be maintained, is allowed to preferably be sent to target spot generation effect.

Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, without departing from the technical principles of the invention, some improvement and modification can also be made, these improvement and modification Also it should be regarded as protection scope of the present invention.

Claims (10)

1. a kind of tumour antigen cyclic peptide or its officinal salt, its cyclic peptide are characterised by：Represented by below general formula (I)：

Wherein X is following formula (Xa), (Xb) or (Xc)；

AA is the natural or alpha-non-natural amino acid of D types or L-type；

N is 1-3.

2. tumour antigen cyclic peptide according to claim 1 or its officinal salt, it is characterised in that wherein AA is leucine, relies ammonia Acid, histidine, tryptophan, aspartic acid or arginine.

3. tumour antigen cyclic peptide according to claim 2 or its officinal salt, it is characterised in that wherein AA is leucine or bad ammonia Acid.

4. tumour antigen cyclic peptide according to claim 1 or its officinal salt, it is characterised in that the cyclic peptide is selected from following Peptide：

5. prepare the method for the tumour antigen cyclic peptide or its officinal salt of claim 1, it is characterised in that comprise the following steps：

I) linear peptides for meeting sequence described in claim 1 are prepared using solid phase or liquid phase synthesizing method；

Ii) using iodine oxidation method, air oxidation process or other suitable oxidation reactions, disulfide bond cyclization is carried out, is obtained down The compound or its salt of formula：

Wherein X and AA is each freely defined in claim 1.

6. tumour antigen cyclic peptide or its officinal salt according to any one of claim 1-4, it is characterised in that pharmaceutically Acceptable salt includes hydrochloride, hydrobromate, sulfate, phosphate, nitrate, acetate, oxalates or tartrate.

7. tumour antigen cyclic peptide according to claim 6, it is characterised in that pharmaceutically acceptable salt is acetate.

8. a kind of pharmaceutical composition, it is characterised in that：Including the tumour antigen cyclic peptide any one of claim 1-4 or its Officinal salt, and pharmaceutically acceptable carrier or excipient.

9. the purposes of tumour antigen cyclic peptide according to claim 1 or its officinal salt in antineoplastic is prepared.

10. purposes according to claim 9, the tumour includes breast cancer, prostate cancer, lymph cancer and lung cancer.