CN105854025A

CN105854025A - Multi-target polypeptide-adriamycin compound and preparation method and application thereof

Info

Publication number: CN105854025A
Application number: CN201610202840.5A
Authority: CN
Inventors: 陈芸; 尤雯; 尤一雯; 盛媛
Original assignee: Nanjing Medical University
Current assignee: Nanjing University; Nanjing Medical University
Priority date: 2016-04-01
Filing date: 2016-04-01
Publication date: 2016-08-17
Anticipated expiration: 2036-04-01
Also published as: CN105854025B

Abstract

The invention provides a multi-target polypeptide-adriamycin compound and a preparation method and application thereof. The method is as below: dissolving polypeptide T10-ERK in a phosphate buffer with pH value of 7.8 to 8.2; then dissolving DOXO-EMCH in a solvent to obtain a solution, wherein the solvent is a mixture of water with N,N'-dimethylformamide; dropwise adding to the DOXO-EMCH solution into a polypeptide solution, and the reacting with stirringat room temperature in the dark; purifying a crude product by an acetonitrile precipitation method, and centrifuging to obtain a refined product of T10-ERK-DOX. The compound has higher tumor growth inhibition characteristic compared to single-targeted drugs. Using a human breast cancer drug-resistance cells MCF-7 / ADR with high expression of a transferrin receptor as a cell model, and a tumor-bearing mice as an animal model, the T10-ERK-DOX can enhance the uptake of adriamycin nutumor cells, increase the intracellular concentration of the drug, reduce the expression activity of ERK, inhibit tumor cell proliferation, reduce P-gp expression, induce apoptosis and inhibit transplant tumor growth in nude mice.

Description

Mutiple Targets polypeptide doxorubicin compound and its preparation method and application

Technical field

The invention belongs to biology and pharmaceutical technology field, be specifically related to a kind of have reverse breast carcinoma resistance Mutiple Targets polypeptide Ah Mycin compound and its preparation method and application.

Background technology

Adriamycin (DOX) belongs to anthracycline compound, is one of maximally effective cancer therapy drug, is widely used in treatment clinically Acute leukemia, breast cancer, lymthoma and various solid tumor.But owing to it is easily generated drug resistance, cause the clinic of adriamycin Application is severely limited.Up to now, the mechanism about drug resistance of tumor cell has a lot, and one of which mechanism is ATP- Binding cassette transporter body (ATP-binding cassette (ABC) transporter) the such as process LAN of P-glycoprotein (P-gp), it can be by Drug efflux, reduces medicine accumulation in cancer cell.Therefore, the most common method of resistance is overcome to be through antagonism at present Tumour medicine is chemically modified the drug efflux avoiding P-gp to mediate.But, increasing evidence shows only by list The modification of one target spot can not good reversing drug resistance because other resistance mechanisms multiple including including Apoptosis inhibitor etc. are same Can promote to form Drug-resistant.

At present, numerous studies show that MAPK signal path and Drug-resistant are closely bound up.ERK is as MAPK cascade reaction One of middle key protein, it can activate transcribing thus promoting that cell grows of downstream gene, play critically important in apoptosis tolerates Effect.Nearest research display phosphorylated CREB (pERK, ERK activated form) expression after DOX stimulates can increase, Consistent with this, mdr cell MCF-7/ADR compares its parent sensitive cells MCF-7, phosphorylation in MCF-7/ADR The expression of ERK is obviously enhanced.Therefore, targeting ERK itself also becomes an attractive treatment concept.Some ERK Inhibitor has also had been introduced into clinical experimental stage.Although ERK micromolecular inhibitor (such as PD098095 and U0126) is Being widely used, but their selectivity receives query, being primarily due to them can occur ATP to depend on other protein kinases The cross reaction relied.Therefore, based on structure design and there is selective peptide inhibitor be developed, wherein polypeptide MPKKKPTPIQLNP, for MAPK/ERK kinase whose N terminal sequence, it can be by affecting the combination of MEK Yu ERK Prevent the activation of ERK, it is thus possible to effectively suppression ERK activity, but it equally exists certain defect, i.e. can not be fine Ground enters intracellular through cell membrane and plays a role.

At present by hybrid peptide and small-molecule drug being coupled together the research forming Mutiple Targets medicine few, one of them reason It is probably the material of the most single chemical constitution when different target spots being played a role simultaneously, respective optimum efficiency can not be reached. Therefore, the design in addition to the effect of overall compound is verified, the work to its corresponding target spot to each part equally With verifying the most respectively, and mass action and each several part effect are carried out the rate of exchange.

Summary of the invention

Solve the technical problem that: it is an object of the invention to provide a kind of Mutiple Targets polypeptide doxorubicin having and reversing breast carcinoma resistance Compound and its preparation method and application.

Technical scheme: Mutiple Targets polypeptide doxorubicin compound, structural formula shown in formula I:

The preparation method of above-mentioned Mutiple Targets polypeptide doxorubicin compound, comprises the following steps: step 1, by polypeptide HAIYPRHGGCGMPKKKPTPIQLNP (T10-ERK) is dissolved in the phosphate buffer of pH value 7.8～8.2, Being dissolved in solvent by the hydazone derivative (DOXO-EMCH) of 6-maleimidocaproyl and obtain solution, described solvent is Water and N, the mixture of N ' dimethylformamide, DOXO-EMCH solution is added dropwise in polypeptide solution, keeps away under room temperature Light stirring reaction；Step 2, uses acetonitrile precipitation method to be purified crude product, prepares highly finished product T10-ERK-DOX after being centrifuged.

Above-mentioned solvent is water and N, the 1:1 volume ratio mixture of N ' dimethylformamide.

Above-mentioned DOXO-EMCH and polypeptide mol ratio are 2:1.

The speed that above-mentioned DOXO-EMCH solution is added dropwise in polypeptide solution is 25 μ L/min.

It is 1.5h that above-mentioned lucifuge is stirred at room temperature the time.

Above-mentioned acetonitrile precipitation method condition is: the volume ratio of acetonitrile and step 1 gained reaction solution is 5:1；After the two mixing, 4 DEG C Place 24h crystallization, so centrifugal that to precipitate, it is product.

The application in preparing antineoplastic of the above-mentioned Mutiple Targets polypeptide doxorubicin compound.

Reversing the medicine of breast carcinoma resistance, active ingredient is the Mutiple Targets polypeptide doxorubicin compound shown in Formulas I.

The application in preparation reverses breast carcinoma resistance preparation of the above-mentioned Mutiple Targets polypeptide doxorubicin compound.

Beneficial effect: 1) T10-ERK of the present invention is polypeptide, chemically synthesizes, good stability；

2) DOXO-EMCH of the present invention is in acidic cancer microenvironment, and hydrazone key selectively disconnects, and discharges pharmacology and lives The adriamycin of property；

3) the 6-maleimidocaproyl hydazone derivative DOXO-EMCH of adriamycin is selected, covalently bound with hybrid peptide, Chemical method is used to prepare Mutiple Targets polypeptide doxorubicin compound；Preparation method is simple, and reaction rate is high, the shortest, produces Purity and the productivity of thing are higher, can be directly used for cell and animal experiment；

4) the Mutiple Targets polypeptide doxorubicin compound of the present invention is combined by each several part, reaches a comprehensive effect, compares Single target drug has higher Tumor growth inhibition feature, with the human breast carcinoma mdr cell of TfR high expressed MCF-7/ADR is cell model, and with tumor-bearing mice as animal model, T10-ERK-DOX can strengthen tumour cell to adriamycin Picked-up, improve drug concentration, reduce ERK expression activity, suppress tumor cell proliferation, reduce P-gp expression, Induce its apoptosis, the growth of suppression nude mice model tumour；

5) the Mutiple Targets polypeptide doxorubicin compound whole structure of the present invention can comprehensive each part (such as T10-DOX, T10-ERK etc.) effect, and will not influence each other between each part, it is possible to make each part play best effective Really.

Accompanying drawing explanation

Fig. 1 is the LC/MS/MS collection of illustrative plates of the T10-ERK-DOX of the present invention；Wherein, the mother of A:T10-ERK-DOX from Subgraph；The daughter ion figure of B:T10-ERK-DOX；

Fig. 2 is that the T10-ERK-DOX and free DOX and T10-DOX of the present invention are to MCF-7/ADR cell growth inhibition Curve；

Fig. 3 is that the picked-up of polypeptide doxorubicin compound and free adriamycin is compared schematic diagram by MCF-7/ADR cell；

Fig. 4 is the Apoptosis situation detection signal that the T10-ERK-DOX and free DOX and T10-DOX of the present invention causes Figure；Wherein, represent DAPI respectively and contaminate core image, the cell image of TUNEL detection apoptosis；

Fig. 5 A is that the part T10-ERK of the T10-ERK-DOX of the present invention affects schematic diagram to what pERK expressed；

Fig. 5 B be T10-ERK cell proliferation speed affect schematic diagram；

Fig. 5 C is after T10-ERK processes, the cell picked-up schematic diagram to DOX；

Fig. 5 D is the T10-ERK impact on cell drug (DOX) sensitiveness；Fig. 5 E is that T10-ERK is to cell P-gp Protein expression affect schematic diagram；

Fig. 6 shows that the T10-ERK-DOX and free trip DOX and T10-DOX of the present invention are to transplantable tumor nude mice model knurl body Long-pending suppression situation schematic diagram.

Detailed description of the invention

Following example further illustrate present disclosure, but should not be construed as limitation of the present invention.Without departing substantially from the present invention In the case of spirit and essence, the amendment that the inventive method, step or condition are made and replacement, belong to the scope of the present invention. If not specializing, the conventional means that technological means used in embodiment is well known to those skilled in the art.

The synthesis of embodiment 1 polypeptide-adriamycin composite and purifying

(1) preparation of T10-ERK-DOX and purifying

Weigh the T10-ERK of 1.90mg, be dissolved in 600 μ L pH 7.8～8.2 phosphate buffers, then weigh 1.08mg DOXO-EMCH, be dissolved in 600 μ L N, in N ' dimethylformamide in water, wherein N, N ' dimethylformamide It is 1:1 with the volume ratio of water, then is added dropwise in polypeptide solution with the speed of 25 μ L/min, room temperature reaction 1.5h, generate T10-ERK-DOX, adds 6mL acetonitrile (reaction solutions of 5 times of volumes), mixing after reaction in solution, 4 DEG C stand 24h, 8000 × g is centrifuged 5min, and precipitation is product, HPLC sample introduction, measures and purifies purity, more than 98%.

The structural formula of T10-ERK-DOX is:

2) LC/MS/MS method and NMR method identify product

LC/MS/MS method identifies product, uses Agilent 1200 series of high efficiency liquid chromatographic system and 6410 triple level Four bar liquid Matter combined instrument, uses Waters xBridge C18 (250mm × 4.6mm, 5 μm) chromatographic column at room temperature to carry out chromatogram and divides From.Mobile phase A is 0.01% formic acid-aqueous solution, and Mobile phase B is 100% acetonitrile；Gradient elution, elution program is: 0～15 Min, 10～90%B；15～20min；90%～90%B；20～25min, 90%～10%B；The ion of mass spectrometer system Source is electron spray ionisation source (ESI)；Cation scan pattern；Spray voltage 4000V；Atomizing pressure 45psi；Atomization temperature 350℃；Atomization air flow 10L/min；Detection mode is full scan (Full Scan) and daughter ion scanning (Product Scan), Acquisition time is located at 200ms；Transmission voltage is set to 120V, and impact energy is set to 30eV, and all data all pass through Agilent MassHunter workstation software (B.01.04 version) gathers and processes.

T10-ERK-DOX parent ion figure is shown in Fig. 1 (A), ESI-MS/MS m/z:1696 [M+2H]²⁺, 1131 [M+3H]³⁺, 848[M+4H]⁴⁺With 679 [M+5H]⁵⁺.When impact energy is set to 30eV, it is seen that ms fragment feature.Such as Fig. 1 (B) Shown in, T10-ERK-DOX contains peptide section sequence specific b ion (m/z 137 (b1), m/z 209 (b2), m/z 322 (b3)), and DOX fragments characteristic (m/z 376), the synthesis of this product can be verified.

Mtt assay measures cell viability assays

By MCF-7/ADR cell dissociation in good condition, it is diluted to 2 × 10 with nutrient solution⁴Cells/mL cell density, blow even after In 96 orifice plates, every hole adds cell suspension 200 μ L, puts 37 DEG C, hatches 24h in 5% carbon dioxide environment so that it is be adherent. Using 200 μ L DOX, T10-DOX and T10-ERK-DOX pastille nutrient solutions instead, each concentration sets 5 parallel holes, simultaneously Blank group is set, is placed in incubator hatching 48h, then adds MTT liquid (5mg/mL) 20 μ L to every hole, continue Continuous cultivation 4h, sucks nutrient solution, adds DMSO 150 μ L, shakes 30min, join at enzyme under room temperature on microwell plate oscillator Each hole light absorption value is measured under immune detector 490nm.With compared with control cells for 100%, with the cell survival of administration group relative to right The percentage of photo cell survival measures its survival rate.

Investigate three kinds of medicines cytotoxic effect to MCF-7/ADR tumour cell by MTT method, draw cell survival Rate curve, calculates IC₅₀Value, evaluates the vitro cytotoxicity of medicine.As in figure 2 it is shown, the IC of DOX and T10-DOX₅₀Value It is respectively 41.5 ± 1.0 μMs and 33.5 ± 1.0 μMs, and the IC of T10-ERK-DOX₅₀It is respectively 20.8 ± 1.1 μMs, explanation Compare DOX and T10-DOX, MCF7/ADR cell more sensitive to T10-ERK-DOX.

The accumulation experiment of intracellular adriamycin

MCF-7/ADR cell is with 1 × 10⁵The density in/hole is inoculated in 6 well culture plates, puts 37 DEG C, 5% carbon dioxide environment In hatch 24h after, add DOX, T10-DOX and T10-ERK-DOX, final concentration of 1.5 μMs, medicine effect 12h. Pastille nutrient solution is abandoned in suction, washes cell 3 times with cold PBS, and fluorescence microscopy Microscopic observation respectively organizes process cell, and result is shown in Fig. 3. MCF-7/ADR cell after different administration processes, the intracellular fluorescence intensity of T10-DOX and T10-ERK-DOX group Apparently higher than DOX group, and T10-ERK-DOX is slightly higher than T10-DOX group.

Nick End mark (Tunel) method detection Apoptosis

By 2 × 10⁷After the cell of/mL density makes cell smear, with 4% paraformaldehyde/PBS liquid, 4 DEG C of fixing 20min.Add The Proteinase K 100 μ L entering 20 μ g/mL is permeabilized, incubated at room 5min, and PBS washs 3 times.Drip 100 μ L Equilibrium liquid, incubated at room 30min, add TdT incubation buffer 20 μ L, after 37 DEG C of incubation 1h, PBS washing, then use DAPI redyes, and analyzes sample under fluorescence microscope, and as shown in Figure 4, DAPI dye core is blueness to result, and apoptotic cell is green.

Apoptosis testing result is as shown in Figure 4.The apoptosis rate of DOX and T10-DOX group is respectively 21.7% and 43.2%, And T10-ERK-DOX group apoptosis rate substantially increases, it is 48.5%.

The T10-ERK impact on the resistance that ERK mediates

MCF-7/ADR cell is respectively exposed to 100 μMs of T10 and T10-ERK 24h being dissolved in serum-free medium, passes through Western bloting detects intracellular pERK and the expression of total ERK.Fig. 5 A shows after T10-ERK is administered, pERK Expression significantly reduce, and ERK expresses unchanged, and T10 group and blank group pERK and total ERK are all without obvious change Change.

T10 and T10-ERK is dissolved in the nutrient solution containing serum respectively, obtains 10 μMs of dosing nutrient solutions, cultivate with this nutrient solution Cell.After cultivating one week, being inoculated in six orifice plates with the every hole of MCF-7/ADR 5 × 10^4 density, every day carries out cell count, Observation of cell growth rate.The most after the administration is complete, to each group of cellular uptake adriamycin ability and the sensitiveness of DOX is entered Row detection (method measures cell viability assays with the accumulation experiment of above-mentioned intracellular adriamycin and mtt assay).Result such as Fig. 5 B With 5C, T10-ERK group cell growth rate slows down, and the picked-up ability of adriamycin is increased.Fig. 5 D shows, through T10-ERK After stimulation, the sensitiveness of DOX is improved by MCF-7/ADR cell, IC₅₀Value is 33.0 ± 1.0 μMs, and T10 and control Group indifference, IC₅₀Value is respectively 38.4 ± 1.0 μMs and 39.7 ± 1.0 μMs.

With above-mentioned 10 μMs of dosing nutrient solutions to MCF-7/ADR cell administration two weeks after, to its P-gp express by cell streaming Instrument detects.Fig. 5 E shows, the P-gp of T10-ERK group cell expresses and reduces, and T10 and blank group there is no substantially Difference.

Internal antitumor activity

The MCF-7/ADR breast cancer cell of exponential phase will be in, with PBS modulation cell suspension to cell number be 1 × l0⁷/ mL, is inoculated in right side of mice armpit with every 0.1mL subcutaneous, sets up mammary carcinoma knurl strain oxter inoculation model.

Inoculate 7-14 days, treat that tumor volume growth is to 50-100mm³, tumor-bearing mice is carried out tail vein injection administration.By lotus knurl Mouse is randomly divided into 4 groups (often group 6), respectively model control group (physiological saline), DOX, T10-DOX and T10-ERK-DOX group, control group tail vein gives physiological saline, and remaining is respectively organized tail vein injection respectively and gives relative medicine, Be administered once every 5 days, successive administration 5 times, dosage be 3mg DOX/kg mouse weight, wherein T10-DOX and T10-ERK-DOX group need to be pressed 3mg DOX/kg mouse weight standard be administered with DOX as active ingredient after conversion again.

After administration, observe the existing state of mouse every day, use vernier caliper measurement gross tumor volume.Treat that each administration group compares with lotus knurl After gross tumor volume between group exists notable difference, terminate to observe.Put to death mouse, claim knurl weight, calculate tumour inhibiting rate.

As shown in Figure 6, DOX, T10-DOX and T10-ERK-DOX group all can suppress the growth of gross tumor volume to a certain extent, Presenting certain antitumor activity, DOX and T10-DOX group tumour inhibiting rate is respectively 45.7 ± 2.8% and 63.0 ± 3.1%, and The average tumour inhibiting rate of T10-ERK-DOX substantially increases (p < 0.05), is 72.2 ± 4.6%.

To sum up, the property that sulfydryl that the double bond on DOXO-EMCH maleimide base group can be free with on polypeptide is covalently bound is utilized Matter, we devise peptide T 10-ERK, with DOXO-EMCH, addition reaction occur, form the many targets of T10-ERK-DOX Putting compound, it is compared single target drug and adds the medicine enrichment at tumour cell, and inhibition cancer cell is bred, and promotes apoptosis, Reduce ERK activity, reduce the expression of P-gp, improve curative effect of medication, reversed Drug-resistant.

Above example only for technology design and the feature of the present invention are described, its object is to allow and is familiar with technique People be to will appreciate that present disclosure and implement according to this, can not limit the scope of the invention with this. All equivalent transformations done according to spirit of the invention or modification, all should contain in protection scope of the present invention Within.

SEQUENCE LISTING

<110> Nanjing Medical University

<120> Mutiple Targets polypeptide-adriamycin composite and its preparation method and application

<130>

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 24

<212> PRT

<213> Artificial sequence

<400> 1

His Ala Ile Tyr Pro Arg His Gly Gly Cys Gly Met Pro Lys Lys Lys

1 5 10 15

Pro Thr Pro Ile Gln Leu Asn Pro

20

Claims

1. Mutiple Targets polypeptide doxorubicin compound, it is characterised in that structural formula shown in formula I:

2. the preparation method of Mutiple Targets polypeptide doxorubicin compound described in claim 1, it is characterised in that comprise the following steps: Step 1, is dissolved in the phosphorus of pH value 7.8～8.2 by polypeptide HAIYPRHGGCGMPKKKPTPIQLNP (T10-ERK) In phthalate buffer, then the hydazone derivative (DOXO-EMCH) of 6-maleimidocaproyl is dissolved in solvent obtain molten Liquid, described solvent is water and N, and DOXO-EMCH solution is added dropwise to polypeptide molten by the mixture of N ' dimethylformamide In liquid, lucifuge stirring reaction under room temperature；Step 2, uses acetonitrile precipitation method to be purified crude product, prepares refined after being centrifuged Product T10-ERK-DOX.

The preparation method of Mutiple Targets polypeptide doxorubicin compound the most according to claim 2, it is characterised in that described solvent is Water and N, the 1:1 volume ratio mixture of N ' dimethylformamide.

The preparation method of Mutiple Targets polypeptide doxorubicin compound the most according to claim 2, it is characterised in that described DOXO-EMCH and polypeptide mol ratio are 2:1.

5. according to the preparation method of claim 2 Mutiple Targets polypeptide doxorubicin compound, it is characterised in that described The speed that DOXO-EMCH solution is added dropwise in polypeptide solution is 25 μ L/min.

The preparation method of Mutiple Targets polypeptide doxorubicin compound the most according to claim 2, it is characterised in that described lucifuge room Temperature mixing time is 1.5h.

The preparation method of Mutiple Targets polypeptide doxorubicin compound the most according to claim 2, it is characterised in that described acetonitrile sinks Shallow lake method condition is: the volume ratio of acetonitrile and step 1 gained reaction solution is 5:1；After the two mixing, place 24h crystallization for 4 DEG C, So centrifugal that to precipitate, it is product.

8. Mutiple Targets polypeptide doxorubicin compound application in preparing antineoplastic described in claim 1.

9. reverse the medicine of breast carcinoma resistance, it is characterised in that active ingredient is the Mutiple Targets polypeptide shown in claim 1 Formulas I Adriamycin composite.

10. the application in preparation reverses breast carcinoma resistance preparation of the Mutiple Targets polypeptide doxorubicin compound described in claim 1.