CN105585615A

CN105585615A - Peptide variants for treating cancer and application of peptide variants

Info

Publication number: CN105585615A
Application number: CN201610002930.XA
Authority: CN
Inventors: 朱凌玮
Original assignee: 朱凌玮
Current assignee: Su Hua Hua
Priority date: 2014-05-29
Filing date: 2014-05-29
Publication date: 2016-05-18
Anticipated expiration: 2034-05-29
Also published as: CN105481949A; CN105601712A; CN105601714B; CN105601714A; CN105601710A; CN105504016A; CN105597078B; CN105601715B; CN105601710B; CN105622722B; CN105601711A; CN105601711B; CN105585615B; CN105601713A; CN105622722A; CN105597078A; CN105481948B; CN105481948A; CN105504017B; CN105504017A

Abstract

The invention relates to peptide variants for treating cancer and an application of the peptide variants. The amino acid sequences of the peptide variants are shown in SEQ ID No:2-15. The peptide variants have higher tumor cell killing effects than wide peptide variants and have wide pharmaceutical prospect.

Description

A kind of peptide variant and application thereof for the treatment of cancer

Technical field

The invention belongs to biological technical field, specifically, the present invention relates to treat the peptide variant of cancer.

Background technology

Microtubule is the constituent of one of three thread networks of cytoskeleton, is complicated, unsettled polymer, micro-The elementary cell of pipe is α-and 'beta '-tubulin dimer. For example, they are responsible for various kinds of cell motion, aixs cylinder transport or motionProperty, particularly, they are provided for forming the stock of mitotic spindle. Microtubule constantly forms different length againHollow tube. Therefore,, by tumoricidal microtubule dynamics, Toplink of the present invention plays and stops the anti-of cancer cell multiplication to haveThe effect (slow down or stop the mitosis of cancer cell) of silk disintegrating agent.

Active peptide of the present invention also shows the plasma membrane that changes specifically cancer cell, and shows at host organismsHealthy cell in do not cause the change that these are identical. Described active peptide has the character of polycation, and therefore they can act onOn the plasma membrane of cancer cell that is rich in negative electrical charge.

In the prior art, in order to improve the activity of albumen or peptide, carry out specificity for the avtive spot of albumen or peptideSudden change and replacement, be conventional way thereby find active higher variant.

Summary of the invention

Peptide shown in sequence SEQIDNo:1 well known in the prior artly can be used to treat cancer. But itsActivity is not very satisfactory. In order further to improve the biologically active of this peptide, applicant is by a large amount of experiments, forIn peptide shown in SEQIDNo:1, point mutation has been carried out and specific modification is carried out respectively at two ends in specific site, therebyObtain the variant itself than the peptide shown in SEQIDNo:1 with higher tumor-suppression activity.

Particularly, be used as the sequence (Table I) as follows of the peptide of the active component for the treatment of cancer:

SEQ sequence number (XA code name)	Sequence	Amino acid number
			1（XA1）	INWLKIAKKVAGML-NH₂	14
2（XA2）	SINWLKIAKKVAGML-NH₂	15
			3（XA3）	SINWLAIAKKVAGML-NH₂	15
4（XA4）	SINWLAIAAKVAGML-NH₂	15
			5（XA5）	SINWLAIAAKVGGML-NH₂	15
6（XA6）	SINWLKIAKKVAGMLL-NH₂	16
			7（XA7）	SINWLKIAKKVAGMLL-NH₂	16
8（XA8）	SINWLAIAKKVAGMLL-NH₂	16
			9（XA9）	SINWLAIAAKVAGMLL-NH₂	16
10（XA10）	SINWLAIAAKVGGMLL-NH₂	16
			11（XA11）	INWLINWLKIAKKVAGML-NH₂	18
12（XA12）	INWLSINWLKIAKKVAGML-NH₂	19
			13（XA13）	INWLSINWLAIAKKVAGML-NH₂	19
14（XA14）	INWLSINWLAIAAKVGGMLL-NH₂	20 1 -->
			15（XA15）	INWLSINWLKIAKKVAGMLL-NH₂	20
16（XA16）	INWL INWLAIAKKVAGML-NH₂	18
			17（XA17）	INWLSINWLAIAKKVAGMLL-NH₂	20

Applicant has confirmation to the research of these peptides of SEQIDNo:2 to SEQIDNo:15, and these peptides are with respect to SEQIDThe peptide of No:1 has higher cytotoxic activity to cancer cell, host's to be treated cell is had to very weak toxicity simultaneouslyOr non-toxic. Active peptide of the present invention changes the microtubule of cancer cell specifically.

The peptide of quoting in Table I is used as treating the medicine of cancer, particularly, can effectively treat leukaemia, liver cancer, glueMatter blastoma etc.

According to another feature of the present invention, produce sequence SEQIDNo:1 to SEQIDNo:15 by chemical synthesisPeptide.

Another object of invention relates to and comprises the pharmaceutical composition of one of defined peptide above.

Advantageously, described composition is waterborne suspension or solution form, or not dressing or the dressing of drying regimeTablet form, for example pill, gel capsule, capsule or powder.

If described pharmaceutical composition is in drying regime, it can for example form sediment with one or more inert diluentsThe mixing such as powder, cellulose, sucrose, lactose or silica. It can also comprise other materials under drying regime, for example a kind ofOr multiple lubricant is as dolomol or talcum powder, colouring agent, dressing (sweet tablet tablet) or varnish.

If pharmaceutical composition of the present invention is liquid form, it can comprise and contains inert diluent for example water, secondPharmaceutically useful solution, suspension, emulsion, syrup and the elixir of alcohol, glycerine, vegetable oil or atoleine. Described drug regimenThing can also comprise other liquid substances except diluent, for example wetting agent, sweetener, thickener, flavor enhancement or stableAgent product.

Described composition optional with pharmaceutically useful nontoxic inert excipient or combination of media, it is characterized in that, can be with skinSkin, transdermal or through skin; Oral; Outside stomach and intestine; Composition described in the form local application of nose or bronchus application.

The pharmaceutical composition of using outward for stomach and intestine can be water-based or non-aqueous solution, suspension or emulsion. These systemsAgent is by particularly for example oil of olive oil and sesame oil, injectable organic ester of the water as solvent or medium, propane diols, vegetable oilAcetoacetic ester or other suitable organic solvents are made. Described pharmaceutical composition can also contain adjuvant, particularly wetting agent, etc.Penetration enhancer, emulsifying agent, dispersant and stabilizing agent.

For local application, advantageously, use described medicine with the form of ointment, emulsifiable paste, gel or patch.

In addition, described pharmaceutical composition can be used alone or combine use with other drug in given treatment, orPerson combines with radiotherapy or operation.

Detailed description of the invention

A)Synthesizing of peptide

Synthetic and the purifying of peptide SEQIDNo:1 to SEQIDNo:17

Described in synthesizing to prepare by N-9-difluorophenyl methoxycarbonyl and NovasynTGS resin manual separation solid phasePeptide. The group of side chain protected comprises the tert-butyl group of serine and the tert-butoxycarbonyl of lysine. There is acetylation in order to prepareThe peptide of N terminal residue, having connected after last amino acid residue, acetic anhydride is joined in resin-peptide complexes, andKeep stirring 1 hour to promote acetylation.

By trifluoroacetic acid/1 that is 10mL.g-1 by concentration, 2-dithioglycol/methyl phenyl ethers anisole/phenol/water (volume ratio82.5:2.5:5:5:5) at room temperature process and within 2 hours, shear described resin-peptide complexes. Filter subsequently to remove treeFat. Then add the ether of 4 ° of C, make thick peptide precipitation, at room temperature after centrifugal 15 minutes, collect thick peptide with 1000g subsequently. SubsequentlyThick peptide is dissolved in water, and uses ShiseidoC-18 semi-preparative column (250mmx10.0mm; 5 μ are m) with containing 0.1%(v/v) 40%(v/v of TFA) carry out chromatography by RP-HPLC under the speed isocratic elution of acetonitrile with 2mL/min. WithUV-DAD detector (ultraviolet PDAD) (Shimadzu, model SPD-M10A) regulates wash-out under 215nm,And the cut of each wash-out is manually collected in the vial of 2mL. Analyze to measure by HPLC and ESI-MS and closeThe sequence of the peptide (two kinds of peptides of acetylation and non-acetylation) becoming. Be synthesized at peptide, after purifying inspection, confirm to have obtained Table I instituteThe peptide sequence showing.

B)The activity of peptide SEQIDNo:1-17 to healthy cell

Two kinds of nononcogenic fibroblast FIBRO1 are contacted with peptide SEQIDNo:1-17 with FIBRO2, healthy thin to observeWhether born of the same parents' microtubule network is also destroyed by this peptide. In the situation that using peptide concentration to be 15 μ M, represent multiple NormocellularHF does not respond (the tubulin density of polymerization does not change) to peptide SEQIDNo:1-15 at all. And SEQIDNo:16-17 process fibroblast larger on the impact of tubulin density, its thickness attenuation about 15%. This saysBright, peptide SEQIDNo:1-15 is to healthy cell non-activity. When peptide SEQIDNo:1-15 is used as mammal, especially peopleTherapeutic agent time, in fact very likely there is little side effect.

C) mensuration of cell proliferation and cell survival rate

Analyze following clone (Table II), to determine the also activity of quantitation of peptides SEQIDNo:1-17.

Table II

Organization type	Clone
		Acute promyelocytic leukemia cell system	NB4
Liver cancer	HepG2
		Glioblastoma	U87M6

Volume with every hole 0.2mL is inoculated into approximately 10000 cells in 96 hole microplates, after 24 hours, uses peptide SEQIDNo:1-17 processes. Under the condition that has peptide SEQIDNo:1-17 by cell at CO₂In incubator, under 37 ° of C, hatch 48Hour. After hatching, by MTT reagent or (3-(4,5-dimethyl-2-yl)-2,5-diphenyl bromination tetrazolium (0.5mg/ml,In the volume of 0.1ml) join in each hole and under 37 ° of C and hatch 2 hours. Then, the contained tetrazole ring of MTT is lived carefullyBorn of the same parents' succinate dehydrogenase reduction. Then, remove supernatant, and add 0.1ml lysis buffer (contain 10%tritonX100 andThe isopropyl alcohol of 10%1NHCl) the bluish violet formazan precipitation that formed to dissolve. After a few minutes, at room temperature use ELIASA to existUnder the detection wavelength of 562nm, measure absorbance. Confirm the survival rate of control cells with the hole that does not contain peptide. Rear thin by processingThe ratio of born of the same parents' absorbance and the absorbance of control cells is assessed the IC50(peptide SEQIDNo:1-17 of each cell at thisConcentration while causing 50% cytotoxicity in a little clone) value, μ M.

Table III

SEQ sequence number (XA code name)	NB4（IC50）	HepG2（IC50）	U87M6（IC50）
				1（XA1）	52.2	35.8	15.41
2（XA2）	37.3	29.3	10.9
				3（XA3）	32.5	27.4	9.8
4（XA4）	34.6	25.4	10.3
				5（XA5）	31.9	26.9	10.1
6（XA6）	40.1	24.8	9.7
				7（XA7）	34.9	28.7	9.6 3 -->
8（XA8）	35.2	27.3	10.0
				9（XA9）	37.8	28.1	10.2
10（XA10）	36.3	25.1	10.4
				11（XA11）	30.9	20.9	9.9
12（XA12）	29.8	19.9	9.3
				13（XA13）	31.7	21.7	9.7
14（XA14）	39.5	22.5	9.7
				15（XA15）	28.7	20.1	10.1
16（XA16）	60.9	45.9	30.5
				17（XA17）	65.3	50.5	35.7

As shown in Table III, peptide SEQIDNo:2-15 has significantly improved the propagation of above three kinds of tumour cells. Than peptide SEQIDNo:1 is reduced significantly to the virose dosage of cancer cell, and SEQIDNo:16 and 17 has improved IC50 value on the contrary, saysBright the application's peptide SEQIDNo:2-15 has the better inhibition of pair tumour cell with respect to prior art, can be furtherFor the associated treatment of tumour.

The drug resistance of cancer cell to peptide

SEQIDNo:1-15 is carried out to drug resistance experiment to cancer cell respectively, and experimental technique is the method for this area routine, knotFruit shows that this peptide has the quick resistance process of not inducing.

Sequence table

<110>Zhu Lingwei

Mono-kind of < 120 > treats peptide variant XA14 and the application thereof of cancer

〈160〉17

〈210〉1

〈211〉14

〈212〉PRT

〈213〉artificialsequence

〈400〉1

INWLKIAKKVAGML

〈210〉2

〈211〉14

〈212〉PRT

〈213〉artificialsequence

〈400〉2

SINWLKIAKKVAGML

〈210〉3

〈211〉15

〈212〉PRT

〈213〉artificialsequence

〈400〉3

SINWLAIAKKVAGML

〈210〉4

〈211〉15

〈212〉PRT

〈213〉artificialsequence

〈400〉4

SINWLAIAAKVAGML

〈210〉5

〈211〉15

〈212〉PRT

〈213〉artificialsequence

〈400〉5

SINWLAIAAKVGGML

〈210〉6

〈211〉16

〈212〉PRT

〈213〉artificialsequence

〈400〉6

SINWLKIAKKVAGMLL

〈210〉7

〈211〉16

〈212〉PRT

〈213〉artificialsequence

〈400〉7

SINWLKIAKKVAGMLL

〈210〉8

〈211〉16

〈212〉PRT

〈213〉artificialsequence

〈400〉8

SINWLAIAKKVAGMLL

〈210〉9

〈211〉16

〈212〉PRT

〈213〉artificialsequence

〈400〉9

SINWLAIAAKVAGMLL

〈210〉10

〈211〉16

〈212〉PRT

〈213〉artificialsequence

〈400〉10

SINWLAIAAKVGGMLL

〈210〉11

〈211〉18

〈212〉PRT

〈213〉artificialsequence

〈400〉12

INWLINWLKIAKKVAGML

〈210〉12

〈211〉19

〈212〉PRT

〈213〉artificialsequence

〈400〉12

INWLSINWLKIAKKVAGML

〈210〉13

〈211〉19

〈212〉PRT

〈213〉artificialsequence

〈400〉13

INWLSINWLAIAKKVAGML

〈210〉14

〈211〉20

〈212〉PRT

〈213〉artificialsequence

〈400〉14

INWLSINWLAIAAKVGGMLL

〈210〉15

〈211〉20

〈212〉PRT

〈213〉artificialsequence

〈400〉15

INWLSINWLKIAKKVAGMLL

〈210〉16

〈211〉18

〈212〉PRT

〈213〉artificialsequence

〈400〉16

INWLINWLAIAKKVAGML

〈210〉17

〈211〉20

〈212〉PRT

〈213〉artificialsequence

〈400〉17

INWLSINWLAIAKKVAGMLL

Claims

1. a peptide that is used for the treatment of cancer, is characterized in that: be XA14, its amino acid sequence is SEQIDNo:14.

2. peptide claimed in claim 1 is as the purposes of medicine.

3. purposes claimed in claim 2, it is used for the treatment of breast cancer, glioblastoma, the cancer of the brain, cervix cancer, knot rectumCancer, cutaneum carcinoma, carcinoma of endometrium, cancer of the stomach, liver cancer and Gastrointestinal Stromal cancer, malignant hematologic disease (leukaemia, Huppert's disease,Lymthoma (Hodgkin's disease, NHL), hepatocellular carcinoma, Kaposi sarcoma, laryngocarcinoma, celiothelioma, the cancer of the esophagus, eyeCancer, osteosarcoma, oophoroma, cancer of pancreas, skin (particularly oral cavity) cancer, lung cancer, prostate cancer, rhabdomyosarcoma, kidney, mammary glandCancer, carcinoma of testis and thyroid cancer, soft tissue cancer, carcinoma of urinary bladder, myeloma (osteocarcinoma) and plasmacytoma.