CN104774245A - Preparation method and use of brain targeting peptide-antineoplastic drug conjugate - Google Patents
Preparation method and use of brain targeting peptide-antineoplastic drug conjugate Download PDFInfo
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- CN104774245A CN104774245A CN201410417617.3A CN201410417617A CN104774245A CN 104774245 A CN104774245 A CN 104774245A CN 201410417617 A CN201410417617 A CN 201410417617A CN 104774245 A CN104774245 A CN 104774245A
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Abstract
The invention discloses a preparation method and use of a brain targeting peptide-antineoplastic drug conjugate. The used brain targeting peptide comprises seven amino acids and has a sequence of FLFRPRN. The desired polypeptide is obtained by a solid phase synthesis method and the conjugate is prepared from the desired polypeptide and an antineoplastic drug by a chemical synthesis method. The invention also provides a use of the conjugate in preparation of brain tumor treatment drugs.
Description
Technical field
The present invention relates to the pharmaceutical field that tumour is relevant, specifically, the present invention relates to a peptide species and antitumour drug coupling forms a kind of novel drugs, this polypeptide itself does not have physiologically active, but the effect had through hemato encephalic barrier, the invention still further relates to preparation method and the application thereof of this polypeptide.
Background technology
The sickness rate of brain tumor and metastatic encephaloma rises year by year.To in its treatment, hemato encephalic barrier is considered to the major cause stoping chemotherapeutics to enter cerebral tissue and then affect the treatment.Hemato encephalic barrier is that body participates in one of internal barrier of inherent immunity, be made up of the brain capillary wall of the pia mater between circulation of blood and brain essence, choroid plexus and the cutose be wrapped in outside wall, can stopped that causal organism and other macromolecular substance enter cerebral tissue and the ventricles of the brain by circulation of blood.Substantially the macromolecular drug of 100%, comprises polypeptide, recombinant protein, monoclonal antibody, all cannot pass through hemato encephalic barrier based on the medicine, gene therapy related drugs etc. of RNA perturbation technique, and the small-molecule drug being greater than 98% also cannot pass hemato encephalic barrier.At present, radiation treatment and chemotherapy can only extend patient vitals 4 months, and therefore the present invention is devoted to research and makes existing medicine better through hemato encephalic barrier.
Polypeptide has polar terminals and non-polar end due to it, therefore has higher availability in vivo, due to itself be endogenous material so toxicity is little, but polypeptide is directly subject to larger restriction as medicine, such as its poor stability, can not be oral.In recent years, polypeptide is as application one of focus becoming new drug development gradually of medicine subsidiary function.Cell-penetrating peptide is the peptide molecule of the class tool specific function starting to recognize mid-term in 20th century, and English academic title is generally written as cell2-penetrating-peptides (CPPs).They are made up of 5 ~ 30 amino acid usually, and typically general have membranes penetration function by 8 ~ 16 Amino acid profiles, also can carry other molecule even supramolecule particle enter in cell.Exactly because this special function, they are counted as the effective intracellular transport instrument of bioactive molecules, are with a wide range of applications.
Polypeptide and medicine formed the example of composition much by covalent manner in the last few years, such as: polypeptide Penetratin and the coupling of Zorubicin covalency are used for the treatment of mammary cancer; Peptide T AT and the coupling of medicine P53 covalency are used for the treatment of cancer eye transfer; Polypeptide YTA4 is coupled with methotrexate covalency and treats mammary cancer.
Summary of the invention
Below detailed description of the present invention:
(1) Brain targeting peptide of the present invention
Brain targeting peptide of the present invention, is made up of 7 amino acid, and aminoacid sequence is SEQ ID NO:1.
(2) preparation method of Brain targeting peptide of the present invention
Polypeptide required for the present invention adopts the method for solid phase to prepare, and solid phase synthesis take solid support resin as carrier, adds amino acid whose process from C end and carboxyl terminal gradually to N end and aminoterminal.
Preferred resin in the present invention: Fmoc-wang resin, 2-cl-trt resin, resin substitution value 0.24 ~ 0.4.
Protected amino acid used in the present invention is: Fmoc-Phe-OH; Fmoc-leu (tBu)-OH, Fmoc-Arg (pbf)-OH, Fmoc-Pro-OH; Fmoc-Asn (trt)-OH, in reaction process, protected amino acid 3 ~ 5 times is excessive.
Deprotecting regent used in the present invention is: piperidines/DMF, and ratio is 10:90 ~ 30:70.
Coupling reagent used in the present invention is: 1. benzotriazole-N, N, N', N'-tetramethyl-urea hexafluorophosphate/I-hydroxybenzotriazole (HOBT), 2. 2-(7-azo benzotriazole)-N, N, N', N'-tetramethyl-urea phosphofluoric acid ester/I-hydroxybenzotriazole (HOBT), 3. N, N'-DIC/I-hydroxybenzotriazole (HOBT).
1. or the 2. plant coupling reagent in the present invention, if use the, so acid binding agent should be added in process in reaction: DIEA/NMP.
The cutting reagent used in the present invention is: TFA/ thioanisole/water/phenol/1，2-ethandithiol, ratio is: 82.5:5:5:5:2.5.
In the present invention use MS-IT-TOF to determine molecular weight, use HPLC purification of crude peptide.
(3) coupling method of Brain targeting peptide of the present invention and antitumor drug Zorubicin
First Zorubicin and anhydride reaction, Doxorubicin molecules builds carboxyl; Again by the carboxyl reaction on the terminal amino group of polypeptide fragment and above-mentioned molecule, generate conjugate; Last separation and purification conjugate.
(4) coupling method of Brain targeting peptide of the present invention and antitumor drug paclitaxel
First taxol and anhydride reaction, taxane molecule builds carboxyl; Again by the carboxyl reaction on the terminal amino group of polypeptide fragment and above-mentioned molecule, generate conjugate; Last separation and purification conjugate.
Accompanying drawing explanation
Accompanying drawing 1: the liquid phase purity spectrogram of targeting peptides
Embodiment
Below in conjunction with embodiment, the present invention is further described in detail, the embodiment provided only in order to illustrate the present invention, instead of in order to limit the scope of the invention.
Below in an example, the various process do not described in detail and method are ordinary methods as known in the art.
embodiment 1: the solid phase synthesis of Brain targeting peptide
Use the solid-phase peptide synthesis (amino acid of use is purchased from Shanghai gill company) of Fmoc strategy, the Liberitity type instrument using CEM company to produce carries out the synthesis of transmembrane polypeptide of the present invention.Working method is carried out according to the instrument specification sheets of manufacturer.
Synthesis agents useful for same is selected:
(1) vector resin: Fmoc-Asn-wang-rink, substitution degree: 0.33.
(2) protected amino acid selected by: Fmoc-Phe-OH; Fmoc-leu (tBu)-OH, Fmoc-Arg (pbf)-OH, Fmoc-Pro-OH; Fmoc-Asn (trt)-OH, in reaction, protected amino acid used 5 times is excessive.
(3) deprotecting regent used in the present invention is: piperidines/DMF, and ratio is 20:80.
(4) coupling reagent used in the present invention is: N, N'-DIC (DIC)/I-hydroxybenzotriazole (HOBT).
(5) cutting reagent used in the present invention is: TFA/ thioanisole/water/phenol/1，2-ethandithiol, ratio is: 82.5:5:5:5:2.5.
HPLC C18 semipreparative column (purchased from Phenomenex company) is used to carry out purifying obtained polypeptide, testing conditions is: determined wavelength: 214nm, mobile phase A: acetonitrile (containing 0.1% trifluoroacetic acid), Mobile phase B: water (containing 0.1% trifluoroacetic acid), elution requirement: by 35%A ~ 50%A in 30 minutes, MS-IT-TOF is utilized to determine gained sterling molecular weight, determine sample purity (liquid phase purity spectrogram is shown in accompanying drawing 1) with HPLC, purity is greater than 95% and obtains polypeptides freeze-dry powder through desalination and lyophilize.
embodiment 2: the conjugate of Brain targeting peptide and antitumor drug Zorubicin
The synthesis of Compound II per
1.0g (1.72mmol) Zorubicin is added, 0.52g (5.20mmol) Succinic anhydried, 30mlTHF in 50m reaction flask, drip 0.66g (5.12mmol) N, N-diisopropylethylamine (DIEA), 27 DEG C are stirred 6h, and solvent evaporated obtains wax, add water stirring, there is yellow solid to separate out, suction filtration, wash twice, drying, obtains yellow solid 1.15g.
The synthesis of Compound I
0.22g (0.32mmol) Compound II per is added, 0.10g (0.31mmol) O-benzotriazole-N, N in 50m reaction flask, N', N'-tetramethyl-urea Tetrafluoroboric acid ester (TBTU), 5mlDMF, nitrogen protection, drip 0.08g (0.62mmol) N, N-diisopropylethylamine (DIEA), after 27 DEG C of stirring 1h, adds 0.10g polypeptide, after 27 DEG C of stirring 3h, termination reaction.Use MS-IT-TOF to determine molecular weight, use HPLC purification of crude product.
embodiment 3: the conjugate of Brain targeting peptide and antitumor drug paclitaxel
The synthesis of compound III
1.50g (1.72mmol) taxol is added, 0.52g (5.20mmol) Succinic anhydried, 30mlTHF in 50m reaction flask, drip 0.66g (5.12mmol) N, N-diisopropylethylamine (DIEA), 27 DEG C are stirred 6h, and solvent evaporated obtains wax, add water stirring, there is yellow solid to separate out, suction filtration, wash twice, drying, obtains yellow solid 1.15g.
The synthesis of compound IV
0.22g (0.32mmol) compound III is added, 0.10g (0.31mmol) O-benzotriazole-N, N in 50m reaction flask; N'; N'-tetramethyl-urea Tetrafluoroboric acid ester (TBTU), 5mlDMF, nitrogen protection; drip 0.08g (0.62mmol) N; N-diisopropylethylamine (DIEA), after 27 DEG C of stirring 1h, adds 0.10g polypeptide; after 27 DEG C of stirring 3h, termination reaction.Use MS-IT-TOF to determine molecular weight, use HPLC purification of crude product.
embodiment 4: conjugate is through the interior evaluating of hemato encephalic barrier
By above-mentioned conjugate I and contrast Zorubicin, conjugate IV and contrast taxol, 10mg/ml solution is configured to physiological saline, by rat (purchased from Chinese Academy of Sciences's Shanghai Experimental Animal Center, SD rat) with etherization, through vena femoralis injection (200 μ L/, 6/group), within latter 30 minutes, get cerebrospinal fluid respectively at injection, and disconnected neck puts to death rat.Cerebrospinal fluid measures the situation through hemato encephalic barrier through LC-MS.
Experimental result shows: conjugate I experimental group can detect Zorubicin in cerebrospinal fluid, and control group does not detect the existence of Zorubicin; Conjugate IV experimental group can detect taxol in cerebrospinal fluid, and control group does not detect the existence of taxol.
embodiment 5: conjugate is through the in-vitro evaluation of hemato encephalic barrier
In the present embodiment, the cell of employing is: Hela, SK-BR-3, NIH3T3 (purchased from Chinese Academy of Sciences's Shanghai life science institute biological chemistry and Institute of Cell Biology), by cell kind in 24 orifice plates, concentration is 2 × 10
5hole, overnight incubation, conjugate I and conjugate IV PBS dissolves, and makes its final concentration be 10umol/l with cell incubation.
In the present embodiment, use immunofluorescence dyeing, 1. use the formaldehyde of 4% 4 ° of lower fixed cells 10 minutes.2. use 3% cold Triton-X100 damping fluid 4 ° of permeation cells 20 minutes.3. the BSA Block buffer of 2% at room temperature closing cell 30 minutes.4. goat-anti people-FLAG mAb at room temperature incubated cell 1 hour.5. 2mg/ml Streptavidin-Alexa499 at room temperature incubated cell 1 hour.Below often walk and all rinse at least twice with PBS.6. 0.3%Triton-X100 damping fluid washes 20 minutes.Observe under the most rearmounted cell and microscope and confocal microscopy.
Experimental result shows: all have dyeing in cell, namely shows that conjugate I and conjugate IV is all by cytolemma.
embodiment 6: conjugate antitumor action is studied
In the present embodiment, the cell of employing is: FCY20201MCF-7, FCY202111590BS524 (T47D), TC-hxbz-286M453, utilizes mtt assay to evaluate the tumor killing effect of conjugate.
Collect logarithmic phase cell, adjustment concentration of cell suspension, every hole adds 100ul, and bed board makes cell to be measured adjust density to 1000-10000 hole.5%CO
2, hatch for 37 DEG C, be paved with (96 hole flat underside) at the bottom of hole, within 4 hours, add the medicine of 5 concentration gradients later to cell monolayer, in principle, every hole 100ul, if 5 multiple holes, continues to hatch 48 hours, observes under inverted microscope.Every hole adds 20ulMTT solution (5mg/ml, i.e. 0.5%MTT), continues to cultivate 4h.Carefully suck nutrient solution in hole, every hole adds 150ul dimethyl sulfoxide (DMSO), puts low-speed oscillation 5min on shaking table, crystallisate is fully dissolved.The light absorption value in each hole is measured at enzyme-linked immunosorbent assay instrument OD490nm place.Zeroing hole (substratum, MTT, dimethyl sulfoxide (DMSO)) is set simultaneously, control wells (the medicine dissolution medium of cell, same concentrations, nutrient solution, MTT, dimethyl sulfoxide (DMSO)).
The present embodiment calculation result adopts formula:
Proliferation inhibition rate=1-(experimental port A value-blank well A value)/(control wells A value-blank well A value), the A value wherein often organized is for deducting the result after withered hole.
Experimental result display: conjugate I and conjugate IV has good tumor killing effect, can calculate conjugate I according to proliferation inhibition rate formula and conjugate IV is respectively 99.99% at the tumour inhibiting rate of 100ug/ml, and 99.90%.99.90% is respectively, 98.16% at the tumour inhibiting rate of 10ug/ml.86.73% is respectively, 79.11% at the tumour inhibiting rate of 1ug/ml.20.98% is respectively, 6.2% at the tumour inhibiting rate of 1ug/ml.
Subordinate list 1: conjugate I absorbance
| 100ug/ml | 10ug/ml | 1ug/ml | 0.1ug/ml | 0.01ug/ml |
| 0.0035 | 0.0123 | 0.1568 | 0.9143 | 1.1192 |
| 0.0038 | 0.0155 | 0.1655 | 0.9212 | 1.1233 |
| 0.0041 | 0.0164 | 0.1673 | 0.9606 | 1.195 |
| 0.0048 | 0.0148 | 0.1695 | 0.9983 | 1.1483 |
| 0.0053 | 0.0199 | 0.1557 | 0.9535 | 1.1355 |
Subordinate list 2: conjugate IV absorbance
| 100ug/ml | 10ug/ml | 1ug/ml | 0.1ug/ml | 0.01ug/ml |
| 0.0037 | 0.0237 | 0.2355 | 1.2022 | 1.2302 |
| 0.0056 | 0.0246 | 0.2652 | 1.2125 | 1.2036 |
| 0.0057 | 0.0302 | 0.2603 | 1.9202 | 1.1902 |
| 0.0056 | 0.0259 | 0.2541 | 1.1608 | 1.1968 |
| 0.0049 | 0.0263 | 0.2545 | 1.1875 | 1.1983 |
Claims (5)
1. a Brain targeting peptide, is characterized in that, described targeting peptides is made up of 7 amino acid, and its aminoacid sequence is SEQ ID NO:1 in sequence table.
2. a conjugate, for Brain targeting peptide according to claim 1 and antitumor drug are formed by chemical bond coupling.
3. a preparation method for conjugate as claimed in claim 2, concrete steps are as follows:
A. the solid-phase peptide synthesis synthesis targeting peptides of Fmoc strategy is used;
B. by antitumor drug and anhydride reaction, drug molecule builds carboxyl;
C. by the carboxyl reaction on the terminal amino group of polypeptide fragment and antitumor drug molecule, amido linkage is built;
D. the separation and purification of conjugate.
4. preparation method according to claim 3, is characterized in that, antitumor drug described in step B is selected from Zorubicin and taxol.
5. conjugate according to claim 2, is characterized in that the application of conjugate in preparation treatment brain tumor medicine.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106674328A (en) * | 2016-12-30 | 2017-05-17 | 天津世传生物科技有限公司 | Polypeptide molecule with brain targeting function as well as preparation method and application thereof |
| CN108101961A (en) * | 2017-12-19 | 2018-06-01 | 东北大学 | A kind of small peptide that can realize brain targeting drug delivery and its application |
| CN112321684A (en) * | 2020-11-03 | 2021-02-05 | 红河学院 | Paclitaxel-antibacterial peptide conjugate, synthesis method and application of paclitaxel-antibacterial peptide conjugate in inhibiting cancer activity |
| CN113925973A (en) * | 2020-07-14 | 2022-01-14 | 辽宁中健医药科技有限公司 | Polypeptide coupled drug, preparation method and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102617852A (en) * | 2012-04-24 | 2012-08-01 | 华东师范大学 | Maleimide-polyglutamic acid-aspartic acid polymer and composite thereof, preparation methods for maleimide-polyglutamic acid-aspartic acid polymer and composite thereof, and application of maleimide-polyglutamic acid-aspartic acid polymer and composite thereof |
| CN103703140A (en) * | 2011-03-30 | 2014-04-02 | 德克萨斯大学系统董事会 | Methods and compositions for targeting adipose cells in mammals |
| CN103874511A (en) * | 2011-06-02 | 2014-06-18 | 阿奇癌症治疗公司 | Brain tumor targeting peptides and methods |
| CN103897043A (en) * | 2012-12-28 | 2014-07-02 | 天津药物研究院 | Preparation and application of transmembrane polypeptide connected lapatinib |
| CN104031119A (en) * | 2014-06-23 | 2014-09-10 | 天津药物研究院 | Brain targeted peptide, and preparation method and application thereof |
-
2014
- 2014-08-22 CN CN201410417617.3A patent/CN104774245A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103703140A (en) * | 2011-03-30 | 2014-04-02 | 德克萨斯大学系统董事会 | Methods and compositions for targeting adipose cells in mammals |
| CN103874511A (en) * | 2011-06-02 | 2014-06-18 | 阿奇癌症治疗公司 | Brain tumor targeting peptides and methods |
| CN102617852A (en) * | 2012-04-24 | 2012-08-01 | 华东师范大学 | Maleimide-polyglutamic acid-aspartic acid polymer and composite thereof, preparation methods for maleimide-polyglutamic acid-aspartic acid polymer and composite thereof, and application of maleimide-polyglutamic acid-aspartic acid polymer and composite thereof |
| CN103897043A (en) * | 2012-12-28 | 2014-07-02 | 天津药物研究院 | Preparation and application of transmembrane polypeptide connected lapatinib |
| CN104031119A (en) * | 2014-06-23 | 2014-09-10 | 天津药物研究院 | Brain targeted peptide, and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| INGALLINELLA P等: "PEGylation of Neuromedin U yields a promising candidate for the treatment of obesity and diabetes", 《BIOORG MED CHEM》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106674328A (en) * | 2016-12-30 | 2017-05-17 | 天津世传生物科技有限公司 | Polypeptide molecule with brain targeting function as well as preparation method and application thereof |
| CN108101961A (en) * | 2017-12-19 | 2018-06-01 | 东北大学 | A kind of small peptide that can realize brain targeting drug delivery and its application |
| CN108101961B (en) * | 2017-12-19 | 2021-01-01 | 东北大学 | Short peptide capable of realizing brain-targeted drug delivery and application thereof |
| CN113925973A (en) * | 2020-07-14 | 2022-01-14 | 辽宁中健医药科技有限公司 | Polypeptide coupled drug, preparation method and application thereof |
| CN112321684A (en) * | 2020-11-03 | 2021-02-05 | 红河学院 | Paclitaxel-antibacterial peptide conjugate, synthesis method and application of paclitaxel-antibacterial peptide conjugate in inhibiting cancer activity |
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