CN101234122A - New purpose of polylysine - Google Patents
New purpose of polylysine Download PDFInfo
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- CN101234122A CN101234122A CNA2008101012689A CN200810101268A CN101234122A CN 101234122 A CN101234122 A CN 101234122A CN A2008101012689 A CNA2008101012689 A CN A2008101012689A CN 200810101268 A CN200810101268 A CN 200810101268A CN 101234122 A CN101234122 A CN 101234122A
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Abstract
The invention discloses a novel use of polylysine, which is an application of L-polylysine or salts acceptable in pharmacy thereof in preparing drugs for preventing and/or curing tumors, in particular in preparing drugs for preventing and/or curing human hepatoma and human colon cancer. The structure of L-polylysine is shown as formula (I). The results of the anti-tumor activity experiment show that L-polylysine has better inhibiting effect on cancer cells. The IC50 of L-polylysine to human hepatoma cell Bel-7402 and human colon cancer cell HCT-8 is 0.76Mu g/ml and 1.35Mu g/ml respectively. The experiment adopts 5-fluorouracil as positive control drug, the IC50 of which to human hepatoma cell Bel-7402 and human colon cancer cell HCT-8 is 0.62Mu g/ml and 1.79Mu g/ml respectively. The anti-tumor activity of L-polylysine is equivalent to that of 5-fluorouracil.
Description
Technical field
The present invention relates to the new purposes of poly-D-lysine.
Background technology
Telomeric dna is to be positioned at end of chromosome and chromosome is had the DNA sequence that is rich in guanine (G) of protective effect, and it is made of one section double-stranded repeat region and one section strand repeat region, and human and mammiferous repetitive sequence unit is TTAGGG.The dna single chain that is rich in G Hoogsteen hydrogen bond between can be by the G base under cationic the inducing forms four serobilas (G4).Studies show that the morbidity of 85～90% malignant tumor is all relevant with the activity of telomerase, and the formation of DNA G4 structure can effectively suppress the activity of telomerase, and then reach the purpose that suppresses growth of tumour cell.Therefore, can inducing DNA G4 form and make it stable chemical compound and be expected to become the antineoplastic lead compound, become the focus of present research.
Studies show that in a large number single stranded DNA (sequence is TTAGGGTTAGGGTTAGGGTTAGGG, abbreviates Hum24 as) is at Na
+Form antiparallel structure G4 in the solution, at K
+Form G4 mixed structure parallel and the antiparallel coexistence in the solution.
Summary of the invention
A kind of new purposes that the purpose of this invention is to provide the L-poly-D-lysine.
To be L-poly-D-lysine or its pharmaceutically acceptable salt prevent and/or treat application in the tumour medicine in preparation to the purposes of L-poly-D-lysine provided by the present invention, and the structural formula of described L-poly-D-lysine is as follows:
Formula (I)
Described L-poly-D-lysine pharmaceutically acceptable salt specifically can be bromine salt.
Described L-poly-D-lysine or its pharmaceutically acceptable salt are particularly suitable for preparation and prevent and/or treat people's hepatocarcinoma and human colon carcinoma medicine.
Described L-poly-D-lysine, its molecular weight are 15000-30000.
Another object of the present invention provides a kind of medicine that prevents and/or treats tumor.
The medicine that prevents and/or treats tumor provided by the present invention, its effective ingredient are the L-poly-D-lysine shown in the following formula (I) or its pharmaceutically acceptable salt:
Formula (I)
Described L-poly-D-lysine pharmaceutically acceptable salt specifically can be bromine salt.
The described tumour medicine that prevents and/or treats is particularly suitable for preparing the medicine that prevents and/or treats people's hepatocarcinoma and human colon carcinoma.
Wherein, the L-poly-D-lysine in the described medicine, its molecular weight are 15000-30000.
The described tumour medicine that prevents and/or treats can import body such as muscle, Intradermal, subcutaneous, vein, mucosal tissue by the method for injection, injection, collunarium, eye drip, infiltration, absorption, physics or chemistry mediation; Or mixed by other materials or wrap up the back and import body.
With L-poly-D-lysine or its pharmaceutically acceptable salt is the antitumor drug of active component, when needing, can also add one or more pharmaceutically acceptable carriers in said medicine.Described carrier comprises diluent, excipient, filler, binding agent, wetting agent, disintegrating agent, absorption enhancer, surfactant, absorption carrier, lubricant of pharmaceutical field routine etc.
Preventing and/or treating tumour medicine and can make various ways such as injection, tablet, powder, granule, capsule, oral liquid, unguentum, cream with the preparation of L-poly-D-lysine or its pharmaceutically acceptable salt.The medicine of above-mentioned various dosage forms all can be according to the conventional method preparation of pharmaceutical field.
The external anticancer activity experiment results of L-poly-D-lysine shows that poly-D-lysine has good inhibitory effect to cancerous cell.The L-poly-D-lysine is to the IC of human liver cancer cell Bel-7402
50Be 0.76 μ g/ml, to the IC of human colon cancer cell HCT-8
50Be 1.35 μ g/ml.The positive control medicine that adopts in the experiment is a 5-fluorouracil, and it is to the IC of human liver cancer cell Bel-7402 and colon cancer cell HCT-8
50Be respectively 0.62 μ g/ml, 1.79 μ g/ml.The active anticancer and the 5-fluorouracil of L-poly-D-lysine are suitable.
The L-poly-D-lysine presses down tumor mechanism experiment result and shows that the L-poly-D-lysine suppresses growth of tumour cell by inducing people and mammal telomeric dna to form parallel G4 structure.
Description of drawings
Fig. 1 is garden two chromatograms of human body telomeric dna Hum24 conformation change under the condition that does not add L-poly-D-lysine and adding L-poly-D-lysine: (a) Hum24; (b) add the L-poly-D-lysine, the mol ratio that makes Hum24 and L-poly-D-lysine is 6: 1; The molar ellipticity of each spectrogram is-6-8 (mdeg).
Fig. 2 induces strand Hum24 for the L-poly-D-lysine and forms the melting point curve figure of parallel G4 structure.
The specific embodiment
Embodiment 1, MTT reducing process detect the experiment of L-poly-D-lysine anti tumor activity in vitro
The ultimate principle of mtt assay is: tetramethyl azo azoles salt [MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] (available from Beijing chemical reagents corporation) is a kind of dyestuff that can accept hydrogen atom.Dehydrogenase relevant with NADP in the living cells mitochondrion can change into xanchromatic MTT insoluble hepatic formazon in cell, dead cell does not then have this function.Behind DMSO dissolving formazon, under certain wavelength, measure optical density value with microplate reader, can quantitatively measure the survival rate of cell.Can calculate growth of tumour cell suppression ratio (%)=(OD according to formula
Contrast-OD
Experiment)/OD
Contrast* 100%, and then calculate half-inhibition concentration (IC
50).
The concrete operations step is as follows:
1) selects the adherent human liver cancer cell Bel-7402 and the human colon cancer cell HCT-8 of exponential phase for use, after 0.25% trypsinization, be mixed with the cell suspension of 5000/ml with the RPMI RPMI-1640 that contains 10% calf serum, be seeded in respectively in 96 well culture plates, 100 μ l are inoculated in every hole, 37 ℃, 5%CO
2Cultivate 24h.
2) establish L-poly-D-lysine group by 4 mass concentration gradients, the 1-4 row hole that 1-3 is capable adds L-poly-D-lysine (molecular weight 15000-30000 from low to high successively by concentration, Sigma) each 10 μ l, being supplemented to every hole final volume with the RPMI RPMI-1640 is 200 μ l, makes that L-poly-D-lysine final concentration is followed successively by 0.005 μ g/ml, 0.05 μ g/ml, 0.5 μ g/ml, 5 μ g/ml in every hole.The positive control drug 5-fluorouracil is equally also established 4 mass concentration gradients, each concentration is with L-poly-D-lysine group, the capable 5-8 row of 1-3 hole adds each 10 μ l of 5-fluorouracil from low to high successively by concentration, being supplemented to every hole final volume with the RPMI RPMI-1640 is 200 μ l, makes that the 5-fluorouracil final concentration is followed successively by 0.005 μ g/ml, 0.05 μ g/ml, 0.5 μ g/ml, 5 μ g/ml in every hole.1-3 is capable, and the 9th row hole is a matched group, adds 200 μ l RPMI RPMI-1640s.1-3 is capable, and the 10th row hole is blank group, and every hole is inoculating cell not, only adds 200ul RPMI RPMI-1640.37 ℃, 5%CO
2Cultivate 3d.
3) abandon supernatant, every hole adds the serum-free medium of the freshly prepared 0.5mg/ml MTT of 100 μ l, and 37 ℃ are continued to cultivate 4h.Carefully abandon supernatant, and add 200 μ l DMSO dissolving MTT formazon precipitation,, on microplate reader, measure the optical density value at wavelength 544nm place with miniature ultrasonic agitator mixing.Calculate the growth of tumour cell suppression ratio according to the following equation, growth of tumour cell suppression ratio (%)=(OD
Contrast-OD
Experiment)/OD
Contrast* 100% (OD wherein
Contrast, OD
ExperimentFor deducting OD
BlankEmpirical value), try to achieve IC by calculating
50Value.Experimental result is as shown in table 1.Experimental result shows: the L-poly-D-lysine is to the IC of human liver cancer cell Bel-7402 and human colon cancer cell HCT-8
50Be respectively 0.76 μ g/ml, 1.35 μ g/ml.The positive control medicine 5-fluorouracil that adopts in the experiment is to the IC of human liver cancer cell Bel-7402 and human colon cancer cell HCT-8
50Be respectively 0.62ug/ml, 1.79ug/ml.Active anticancer and 5-fluorouracil that the L-poly-D-lysine is described are suitable.
Table 1.L-poly-D-lysine cytotoxicity test result
| The sample title | Concentration (μ g/ml) | Human hepatoma cell strain (Bel-7402) | Human colon cancer cell strain (HCT-8) | ||||
| OD value (meansigma methods) | Suppression ratio (%) | IC 50 (μg/ml) | OD value (meansigma methods) | Suppression ratio (%) | IC 50 (μg/ml) | ||
| The L-poly-D-lysine | 5 | 0.4414 | 79.07 | 0.76 | 0.6055 | 69.75 | 1.35 |
| 0.5 | 2.2356 | -5.98 | 1.9319 | 3.49 | |||
| 0.05 | 1.9888 | 5.72 | 1.8701 | 6.57 | |||
| 0.005 | 2.0387 | 3.35 | 2.0061 | -0.22 | |||
| 5-fluorouracil | 5 | 0.3589 | 82.71 | 0.62 | 0.6451 | 68.39 | 1.79 |
| 0.5 | 1.2239 | 41.04 | 1.8457 | 9.57 | |||
| 0.05 | 2.2089 | -6.41 | 2.1224 | -3.98 | |||
| 0.005 | 2.0727 | 0.16 | 2.1373 | -4.71 | |||
| Contrast | 2.1094 | 2.0017 | |||||
Under the condition that non-metallic ion exists, Hum24 is the existence of strand configuration in the solution, and this configuration has obvious characteristics in garden two chromatographs (CD): a posivtive spike occurs near 255nm, a negative peak occurs near 238nm.When adding the finite concentration poly-D-lysine in the strand Hum24 solution, Hum24 is converted into a kind of parallel G4 structure by strand, has obvious characteristics to be in the CD spectrum: a posivtive spike occurs near 260nm, a negative peak occurs near 240nm.
Concrete experimental technique is as follows: buffer solution is the aqueous solution of being made up of the material of following final concentration: 10mMTris-HCl, 1mM EDTA; PH 7.4.Obtain the Hum24 mother solution that concentration is 200 μ M in the buffer solution as described in 30OD strand Hum24 (the handsome Bioisystech Co., Ltd in Shanghai) (its sequence is shown in sequence in the sequence table 1) is dissolved in.(molecular weight 15000-30000 Sigma) is dissolved in that to obtain concentration in the described buffer solution be 200 μ M L-poly-D-lysine mother solutions with 3.8mg L-poly-D-lysine.L-poly-D-lysine mother solution is diluted to concentration with described buffer solution is respectively 0.2 μ M, 0.25 μ M, 0.33 μ M, 0.4 μ M, 0.5 μ M, 0.7 μ M, 1 μ M, 2 μ M L-Poly-L-Lysine Solutions.The strand Hum24 solution of getting 200 μ M adds the L-Poly-L-Lysine Solution of above-mentioned concentration respectively, makes that the mol ratio of strand Hum24 and L-poly-D-lysine was respectively 10: 1 in the mixed solution, and 8: 1,6: 1,5: 1,4: 1,3: 1,2: 1,1: 1.With above-mentioned mixed solution, all hatch 12h at 25 ℃, adopt the circular dichroism spectrometer to measure the situation of change of its conformation.Experimental result shows that strand Hum24 is extremely important for the formation of parallel G4 structure with the mol ratio of L-poly-D-lysine, is 8 in its mol ratio only: 1-4: could form parallel G4 structure at 1 o'clock.Wherein, strand Hum24 and L-poly-D-lysine mol ratio are that garden two chromatograms of the parallel G4 structure that formed in 6: 1 o'clock are shown in b among Fig. 1.
To be that the parallel G4 structure that formed in 6: 1 o'clock is carried out Measurement of melting point by circular dichroism spectra alternating temperature determination experiment with L-poly-D-lysine mol ratio with strand Hum24.The instrument that adopts is a Jasco815 circular dichroism spectrometer, and the CD absorption cell light path that adopts in the CD experiment is 1cm.Before carrying out the CD experiment, use high-purity N
2Deoxygenation 5 minutes, and also use high-purity N in the experiment always
2To guarantee not having ozone to occur, before the CD experimental data is gathered, carry out the baseline check and correction as protection gas with buffer solution.Gather CD spectrum near ultraviolet band 220nm-320nm, scanning speed is 500nm/min, and times of collection is 4 times.Adopt the JascoPTC-423S temperature control instrument in the alternating temperature experiment, programming rate is 2 ℃/min, carries out the G4 Measurement of melting point by monitoring 262nm CD signal, and the result as shown in Figure 2.The fusing point that records parallel G4 structure is 45 ℃.
Illustrate that the L-poly-D-lysine forms the growth that parallel G4 structure suppresses tumor cell by inducing people and mammal telomeric dna.
Sequence table
Claims (10)
1, the L-poly-D-lysine shown in the formula (I) or its pharmaceutically acceptable salt prevent and/or treat application in the tumour medicine in preparation, and the structural formula of described poly-D-lysine is as follows:
Formula (I)
2, application according to claim 1 is characterized in that: described L-poly-D-lysine pharmaceutically acceptable salt is a bromine salt.
3, application according to claim 1 and 2 is characterized in that: described tumor behaviour hepatocarcinoma.
4, application according to claim 1 and 2 is characterized in that: described tumor is a human colon carcinoma.
5, according to the arbitrary described application of claim 1 to 4, it is characterized in that: the molecular weight of described L-poly-D-lysine is 15000-30000.
6, a kind of medicine that prevents and/or treats tumor, its effective ingredient are the L-poly-D-lysine shown in the following formula (I) or its pharmaceutically acceptable salt:
Formula (I)
7, medicine according to claim 6 is characterized in that: described L-poly-D-lysine pharmaceutically acceptable salt is a bromine salt.
8, according to claim 6 or 7 described medicines, it is characterized in that: described tumor behaviour hepatocarcinoma.
9, according to claim 6 or 7 described medicines, it is characterized in that: described tumor is a human colon carcinoma.
10, according to the arbitrary described medicine of claim 6 to 9, it is characterized in that: the molecular weight of described L-poly-D-lysine is 15000-30000.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101012689A CN101234122B (en) | 2008-03-03 | 2008-03-03 | New purpose of polylysine |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101012689A CN101234122B (en) | 2008-03-03 | 2008-03-03 | New purpose of polylysine |
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| Publication Number | Publication Date |
|---|---|
| CN101234122A true CN101234122A (en) | 2008-08-06 |
| CN101234122B CN101234122B (en) | 2010-06-30 |
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ID=39918189
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102048694B (en) * | 2009-11-06 | 2013-03-13 | 复旦大学 | Polypeptide-modified liver tumor-targeted nano medicine delivery system and preparation method thereof |
| CN110870914A (en) * | 2018-08-31 | 2020-03-10 | 成都夸常奥普医疗科技有限公司 | Use of amino acid nutrients and pharmaceutical compositions containing same |
| CN110870918A (en) * | 2018-08-31 | 2020-03-10 | 成都夸常奥普医疗科技有限公司 | Pharmaceutical composition containing amino acid nutrients and antitumor chemotherapeutic drugs and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| HU202553B (en) * | 1987-10-21 | 1991-03-28 | Mta Kutatas Es Szervezetelemzo | Process for producing isopolypeptides composed of diamino monokarboxylic acids and pharmaceutical compositions comprising same, as well as plant protective comprising polyisolysine |
| JP4342761B2 (en) * | 2001-04-17 | 2009-10-14 | 花王株式会社 | Disinfectant composition |
-
2008
- 2008-03-03 CN CN2008101012689A patent/CN101234122B/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102048694B (en) * | 2009-11-06 | 2013-03-13 | 复旦大学 | Polypeptide-modified liver tumor-targeted nano medicine delivery system and preparation method thereof |
| CN110870914A (en) * | 2018-08-31 | 2020-03-10 | 成都夸常奥普医疗科技有限公司 | Use of amino acid nutrients and pharmaceutical compositions containing same |
| CN110870918A (en) * | 2018-08-31 | 2020-03-10 | 成都夸常奥普医疗科技有限公司 | Pharmaceutical composition containing amino acid nutrients and antitumor chemotherapeutic drugs and application thereof |
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| CN101234122B (en) | 2010-06-30 |
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