CN104000835B

CN104000835B - P glycoprotein inhibitors

Info

Publication number: CN104000835B
Application number: CN201310060453.9A
Authority: CN
Inventors: 赵瑞芝; 冯丽敏; 王银洁; 陈振兴; 余靖宏; 史长灿; 肖志偲
Original assignee: Guangdong Hospital of Traditional Chinese Medicine
Current assignee: Guangdong Hospital of Traditional Chinese Medicine
Priority date: 2013-02-26
Filing date: 2013-02-26
Publication date: 2016-03-23
Anticipated expiration: 2033-02-26
Also published as: CN104000835A

Abstract

The invention discloses a kind of P glycoprotein inhibitors, in vitro tests finds, saikosaponin C has the significant effect suppressing P glycoprotein activity, effectively can improve the picked-up of HEK293 cell for P glycoprotein substrate colchicine, compared with classical P glycoprotein inhibitors verapamil, effect maintains an equal level; Toxicity research shows, and the toxicity of saikosaponin C is very little, does not affect the propagation of HEK293 cell; Mechanism Study shows, saikosaponin C significantly can suppress the expression of P glycoprotein, and therefore, described saikosaponin C can be used as P glycoprotein inhibitors.P glycoprotein inhibitors-saikosaponin C of the present invention, although inhibition is compared with classical P glycoprotein inhibitors verapamil, effect is slightly low, and its toxicity is very little, does not affect the propagation of HEK293 cell, has potential clinical value.

Description

P glycoprotein inhibitors

Technical field

The present invention relates to a kind of P glycoprotein inhibitors, be specifically related to the application of saikosaponin C as P glycoprotein inhibitors.

Background technology

Cancer therapy drug drug resistance causes chemotherapy failure, reduces the reason of curative effect of medication, is also one of Major Difficulties of facing of current cancer chemotherapy.P glycoprotein be generally acknowledge at present cause one of arch-criminal of chemotherapy failure, finding P glycoprotein inhibitors, is one of focus of tumor research to improve chemotherapeutical medicine curative effect.

P glycoprotein is defined first in document " JulianoRL; LingV.AsurfaceglycoproteinmodulatingdrugpermeabilityinCh inesehamsterovarycellmutants.Biochim.Biophys.Acta.; 1976,455:152-162. ".P glycoprotein also known as Mdr-p 1 or ATP in conjunction with box protein 1, at document " MDean, YHamon, GChimini.ThehumanATP-bindingcassette (ABC) transportersuperfamily.Journaloflipidresearch, 2001, 42:1007-1017. " and " AlfredH.Schinkel, JohanW.Jonker.MammaliandrugefluxtransportersoftheATPbind ingcassette (ABC) family:anoverview.AdvancedDrugDeliveryReviews, 2012, 64:138-153. " in have detailed report, be a class energy dependence transport protein, compound antiports different for many structures can be gone out cell.

Summary of the invention

The object of the invention is open a kind of P glycoprotein inhibitors, to meet the needs of clinical practice.

In vitro tests finds, saikosaponin C has the significant effect suppressing P glycoprotein activity, effectively can improve the picked-up of HEK293 cell for P glycoprotein substrate colchicine, can be used for preparing the medicine suppressing P glycoprotein activity; Compared with classical P glycoprotein inhibitors verapamil, effect maintains an equal level; Toxicity research shows, and the toxicity of saikosaponin C is very little, does not affect the propagation of HEK293 cell; Mechanism Study shows, saikosaponin C significantly can suppress the expression of P glycoprotein, and therefore, described saikosaponin C can be used as P glycoprotein inhibitors.

HEK293 cell, can obtain from U.S.'s strain classical collection center (ATCC), and with introducing in detail and explanation.

Described saikosaponin C, its chemical constitution is as follows:

Its preparation method, can see document " Zhou Shouli. the preparation of saikoside a, c, d reference substance. practical medical technologies magazine, 2005,12 (9): 2387-2388. " report;

Its physicochemical property is: Molish reacting positive, and Lieberman-Burchard reaction produces purple.Soluble in water, rare alcohol, particularly hot water and hot alcohol, in butanols and amylalcohol, dissolubility is large, indissoluble or be insoluble to benzene, ether, chloroform equal solvent.Saikosaponin C can adopt commercially produced product, as above the product of Hiroad standing grain Pharmaceutical Technology Co., Ltd.

The dosage of P glycoprotein inhibitors of the present invention is 20mg ~ 200mg every day, and (experiment in vitro), specifically according to the concrete condition of patient, can be determined by doctor, puts on the patient needing treatment by oral route;

The invention still further relates to a kind of pharmaceutical composition, comprise the treatment saikosaponin C of effective dose and pharmaceutically acceptable carrier, described carrier as starch, lactose, methylcellulose, the pharmaceutic adjuvants such as ethyl cellulose, hydroxypropyl emthylcellulose, sucrose.

P glycoprotein inhibitors-saikosaponin C of the present invention, although inhibition is compared with classical P glycoprotein inhibitors verapamil, effect is suitable, and its toxicity is very little, does not affect the propagation of HEK293 cell, has potential clinical value.

Detailed description of the invention

Embodiment 1

Saikosaponin C is to the research of normal rat hepatocytes picked-up colchicine activity influence:

HEK293 cell is inoculated in six orifice plates, removes culture medium after 2d, is divided into blank group, saikosaponin C group and verapamil group; Blank group cell does not add process, the saikosaponin C group complete medium verapamil group added containing the saikosaponin C of 50 μMs of colchicine and 100 μ g/m then adds complete medium containing 50 μMs of verapamils; Final volume is fixed as 2ml, cultivates after 48 hours, with PBS buffer solution for cleaning cell once; The each group of culture medium added containing 50 μMs of colchicine, places in 37 DEG C of incubators and continues to hatch 60 minutes.After by cell harvesting in centrifuge tube, discard pastille culture medium in hole, the PBS adding pre-cooling cleans 1 time, centrifugal supernatant discarded; Add the pure water of 200 μ l in every solencyte, vortex mixes, and frozen-thawed cell suspension 1 time, the then centrifugal 15min with 14000 × g, moved to by supernatant in new pipe ,-80 DEG C save backup.Above all operations, except specified conditions, all carries out in ice bath.Each experiment repetition 3 times.

Component and the content of culture medium are as follows:

DMEM high glucose medium 89%

Hyclone (FBS) 10%

Green grass or young crops-streptomycin dual anti-1%

DMEM culture medium is the abbreviation of Dulbecco ' sModifiedEagleMedium, is divided into again high glycoform and low-sugar type simultaneously; Adopt high glycoform DMEM culture medium herein, containing 4500mg/LD-glucose and L-glutaminate, not containing Sodium Pyruvate.See the report of document " DulbeccoR, FreemanG.PlaqueProductionbythePolyomaVirus.Virology, 1959,8:396-397. ", can adopt commercially produced product, if the Invitrogen company trade mark is the product of C11995.

DMEM high glucose medium composition is as follows:

Accurate absorption culture fluid sample 175 μ l, then add the methanol vortex mixing of triplication, after the centrifugal 15min of 15000g, Aspirate supernatant, in a new EP pipe, volatilizes, then melts again with 175 μ l mobile phases, gets supernatant loading and measure after the centrifugal 15min of 15000g.Chromatographic condition DiamonsilC ₁₈post (250 × 4.6mm, 5 μm), mobile phase is methanol-water (55:45), and flow velocity is 1ml/min, and column temperature is 25 DEG C, and determined wavelength is 353nm, and sample size is 10 μ l.

The results are shown in Table 1.

Table 1 saikosaponin C is on the impact (n=3) of HEK293 cellular uptake colchicine

Note: * represents and to compare with matched group, and difference has statistical significance, P < 0.01; * represents and to compare with matched group, and difference has statistical significance, P < 0.05.

Note: P glycoprotein is present in Cell protection on cell membrane " entrance guard ", determines which material can enter in cell, the content of the colchicine that applicant measures is intracellular level, and intracellular level raises the activity inhibited of prompting P glycoprotein.

Embodiment 2

Saikosaponin C is on the impact of HEK293 cell P P-glycoprotein expression

1. the extraction of total protein of cell

Cell is inoculated in 100 × 20mm culture dish, adds each concentration Radix Bupleuri (processed with vinegar) component and hatch 48h altogether when cell reaches exponential phase; Separately establish matched group (not adding drug treating).Then culture medium in culture dish is discarded, add the PBS buffer solution for cleaning twice of pre-cooling, by cell harvesting in centrifuge tube; Centrifugal, supernatant discarded.Add 100 μ lRIPA lysates by every ware cell, repeatedly lash with 1ml syringe and make cracking complete; In 4 DEG C, the centrifugal 15min of 12000g, moved to by supernatant in new EP pipe ,-80 DEG C of Refrigerator stores are for subsequent use.All operations all carries out on ice bath, each experiment repetition 3 times.

Component and the content of culture medium are as follows:

DMEM high glucose medium 89%

Hyclone (FBS) 10%

Green grass or young crops-streptomycin dual anti-1%

2 protein immunoblots

The preparation of 2.1 loading samples: after measuring each sample protein content by BCA method, sample protein amount is made to be 200 μ g, add 5 × sample-loading buffer and 2.5 μ l20 × DTT reducing agents of 10 μ l, and add appropriate water and supply volume to 50 μ l, in 95 DEG C of heat denatured 10min after vortex mixing, place-20 DEG C and save backup.

2.2 gel electrophoresiss: select the SDS-PAGE gel of 6%, Tris-glycine-SDS electrophoretic buffer, first with 70V electrophoresis 30 minutes after loading, then move to gel end with 100V electrophoresis to indicator.

2.3 transferring films:

(1) film process: cut out the 0.45 μm pvdf membrane onesize with gel in advance, infiltrates to translucent with methanol, takes out the cleaning of use water for several times, to immerse in Tris-glycine transferring film buffer more than 20 minutes together with filter paper.

(2) transferring film: membrane-transferring device installs by the order of negative electrode carbon plate, pad, filter paper, gel, pvdf membrane, filter paper, pad, carbon anode plate from bottom to up successively, alignment, successively removes bubble, fixes to clamp, put into transferring film groove, switch on power; With 100V voltage transferring film 3h.

2.4 immunoreation:

(1), after transferring film completes, first TBS-Tween20(TBST is used) buffer washes film 3 times, each 5 minutes; Add the TBST buffer containing 5% defatted milk powder, room temperature steadily shakes 1h.Mouse monoclonal P-gp primary antibodie (1:500) is added, 4 DEG C of overnight incubation after discarding confining liquid.Within second day, moved to that room temperature water yawing is dynamic hatches 3h, abandon primary antibodie, TBST washs 3 times, each 15 minutes; Add that anti-(1:3000) room temperature water yawing of Goat polyclonal anti-mouse two is dynamic hatches 2h, TBST washs 3 times, each 15 minutes.Add appropriate ECL working solution and film reaction 5 minutes, develop and print film.

(2) antibody combined with RestoreWesternBlotStripping buffer solution elution pvdf membrane 5 minutes, TBST washs 3 times, each 5 minutes, 5% defatted milk powder-TBST buffer blind 30 minutes.All the other operations are the same, add Pan-cadherin mono-antibody (1:3000) and goat-anti rabbit two anti-(1:3000).

(3) gray value of each swimming lane P-gp and Pan-cadherin albumen in film is drawn by Bio-radGelDocXR imaging system, the ratio of calculating and P-gp/Pan-cadherin; Again by it compared with matched group gray value, draw gray scale ratio.The results are shown in Table 2

Table 2 saikosaponin C is on the impact of HEK293 cell P P-glycoprotein expression

Note: * represents that difference has statistical significance compared with Pgp-CTRL group cell, P < 0.05.

Table 2 shows, saikosaponin C can lower the expression of P glycoprotein in HEK293 cell.

Embodiment 3

Saikosaponin C is on the impact of HEK293 cytoactive

With 12500cells/cm ²density is inoculated in HEK293 cell and 96 orifice plates, makes every hole final volume be 100 μ l, is siphoned away by complete medium, add the pastille culture medium of series concentration, OD value after improved MTT Colorimetric Assay dosing 48h when inoculating 2 days.Calculate medicine to the growth inhibition ratio of cell and half-inhibition concentration IC ₅₀value, formula is as follows:

(1) inhibitory rate of cell growth IR=1-(OD _{570(pastille)}-OD _{570(is blank)})/(OD _{570(contrasts)}-OD _{570(is blank)}), OD _{570(pastille)}and OD _{570(contrasts)}the cell sample being respectively dosing and blank culture fluid in MTT experiment in the absorption value at 570nm place, OD _{570(is blank)}for the absorption value of culture fluid at 570nm place in zeroing hole.

Dissolubility due to saikosaponin C and fatty acid is maximum only can reach 1mg/ml and 100 μ g/ml; By saikosaponin C-1000,100,10,1,0.1 μ g/ml, deliver medicine to the HEK293 cell of same condition.Find that saikosaponin C cell growth does not play inhibitory action, (average rate of increase is respectively: 41.9%, 108.1%, 58.0%, 20.8%, 20.0%) to promote the growth of cell on the contrary.

This experimental result prompting saikosaponin C is safe for cell.

Component and the content of culture medium are as follows:

DMEM high glucose medium 89%

Hyclone (FBS) 10%

Green grass or young crops-streptomycin dual anti-1%.

Embodiment 4

Saikosaponin C 50mg

Lactose 200mg

Fluidizer 10mg

Can be made into tablet, capsule, granule etc.

Claims

1. saikosaponin C suppresses the application in P glycoprotein activity medicine in preparation.

2. application according to claim 1, is characterized in that, described suppression P glycoprotein activity medicine comprises the saikosaponin C for the treatment of effective dose and pharmaceutically acceptable carrier.