CN102533927A - Method for building drug-oral-taking absorption forecasting and screening models - Google Patents
Method for building drug-oral-taking absorption forecasting and screening models Download PDFInfo
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- CN102533927A CN102533927A CN2012100231420A CN201210023142A CN102533927A CN 102533927 A CN102533927 A CN 102533927A CN 2012100231420 A CN2012100231420 A CN 2012100231420A CN 201210023142 A CN201210023142 A CN 201210023142A CN 102533927 A CN102533927 A CN 102533927A
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Abstract
The invention relates to a method for building drug-oral-taking absorption forecasting and screening models. On the basis of a 21-day Caco-2 cell model built in early days forecasting compound absorption, a forecasting method of a 7-day Caco-2 cell model is further built, optimized and verified, and the testing index and the cell morphology of the model are optimized and verified, so that a most fitting Caco-2 cell model with drug-oral-taking absorption and high throughput screening is obtained. According to the method, the culture time of the cell model can be greatly shortened, so that the possibility of appearing pollution in the 21-day long-term culture of cells can be greatly reduced, the labor strength is reduced, and the oral-taking absorption forecasting and screening of a lot of lead compounds existing today are responded.
Description
Technical field
The present invention relates to the medicaments sifting model field, be specifically related to the establishment method that drug oral absorbs prediction and screening model, can be applicable to prediction and screening that drug oral absorbs.
Background technology
Caco-2 cell (the human colon carcinoma cell line, human colon adenocarcinoma cell line are called for short Caco-2) derives from human colon cancer cell, is to generally acknowledge at present to be used to predict one of external model that the interior drug absorption of human intestinal is the most classical.The Caco-2 cell can spontaneously under culture condition carry out the differentiation of epithelium appearance and can form being tightly linked; Differentiate villous surface and basal surface; The functional expression of its morphology, marker enzyme, penetration signature etc. are similar with intestinal epithelial cell; Therefore, this kind cell can be simulated intestinal epithelial cell, is widely used in the research of physics and biochemical barrier in the drug absorption process.
Caco-2 can express many carrier proteinss and multiple drug metabolism enzyme in traditional Caco-2 culture model, comprise P-gp (P-glycoprotein, P-gp), CYP3A4 etc.The establishment method of traditional C aco-2 cell model is: on basic medium, cultivated 21 days behind the cell kind plate, changed liquid every other day, and changed liquid back 14 day every day in preceding 7 days; But the apparent in view defective of this model is to reach 21 days incubation time, makes it be applied to predict that the experimental period when drug oral absorbs is long, can not deal with a large amount of now lead compounds and emerge in large numbers the required high flux screening in back.
Summary of the invention
The objective of the invention is defective to traditional longer incubation time of Caco-2 culture model needs; Provide a kind of drug oral to absorb the establishment method of prediction and screening model; Shorten cell culture period to greatest extent, improve the efficient that drug oral absorbs prediction and screening.
We's ratio juris is to utilize external model and the systemic dependency of body, carries out the prediction that oral pharmaceutical absorb.
The present invention realizes above-mentioned purpose through following technical scheme.
A kind of drug oral absorbs the establishment method of prediction and screening model, comprises the steps:
(1) is base culture base Caco-2 cell with DMEM (Dulbecco ' s modified Eagle medium), contains the foetal calf serum of volume(tric)fraction 20%, 1% non-essential amino acid, 100 UmL in the basic medium
-1Penicillium mould and 100 UmL
-1Streptomycin sulphate;
(2) the kind plate promptly adds mouse tail collagen, air dried overnight with mouse tail collagen bed board previous day in each hole on the Transwell plate; The compound method of mouse tail collagen is: gets the 100ml ultrapure water, adds 100 μ l acetate, and autoclaving, the cooling back adds 5mg/ml mouse tail collagen 2ml, and with the methyl alcohol dilution, the dilution volume ratio is a methyl alcohol: mouse tail collagen=4:1 before using;
(3) when the Caco-2 cell grows to 80%～90% at the bottom of covering the culturing bottle bottle, trysinization, on the Transwell plate, Transwell plate cavity of resorption adds basic medium with the Caco-2 cell inoculation;
Change basic medium (4) the 2nd, 3 day every day, added the differentiation substratum on the 4th, 5,6 day, division culture medium makes for interpolation MIOT after adopting epithelial cell differentiation basic medium; After cultivating 6 days altogether, obtain to form the Caco-2 cell of tight individual layer.
Preferably, cultivating the Caco-2 cell in the step (1) is that the Caco-2 cell is put into culturing bottle, places in the incubator and cultivates, and changes basic medium every other day; The culture condition of incubator is: 37 ℃ of temperature feed 5% CO
2, relative humidity 90%.
Preferably, add above-mentioned mouse tail collagen 800 μ L in the step (2) in each hole.
Preferably, the inoculum density of step (3) is 5 * 10
5/ hole, Transwell plate cavity of resorption adds basic medium 1.5 mL.
Wherein the Transwell plate is a kind of carbonic acid polyester film with 0.3 μ m aperture, works to support the Caco-2 monolayer; MIOT in the step (4) is MIOT (BD) Company products, is to comprise mixture of ingredients such as Sigma I8405, cholera rhzomorph.
Before the transport experiment of medicine is carried out and after finishing; All measure transepithelial cell resistance (transepithelial electrical resistance with the cell potential appearance; TEER), to confirm the compactness and the integrity of monolayer, qualified cell can be used for experiment.
The present invention compared with prior art has following advantage:
A) effective screening rate of the external absorption of raising medicine.Model of the present invention is got it filled and is measured after making medicine through processes such as the absorption of cell, metabolism, makes result and integral experiment data identical basically of in-vitro screening; On the other hand, the present invention has reduced the incubation time of cell, thereby has reduced the labile factor in the experiment, as polluting etc.Therefore this quick medicament screening model has improved the safety of drug absorption prediction and screening, can improve the screening rate of medicine effectively;
B) simplified screening procedure.Conventional external Caco-2 cell screening model need reach 21 days cultivation, the efficient that this has restricted the external transhipment of initial stage lead compound greatly and has absorbed screening.This invention is reduced to incubation time 6 days, and breakneck acceleration is more than 3 times of traditional incubation time, has simplified screening procedure, has practiced thrift lot of manpower and material resources;
C) easy to operate, practice thrift cost.Contrast is the screening model of report both at home and abroad, and the economic benefit of model of the present invention is higher.About the model of rapid screening, report is arranged with fiber collagen bed board, but its cost is higher, the conventional model that consumption rate was cultivated in 21 days on the cost is more very.The present invention has utilized mouse tail collagen bed board, equally also can be effective, and has realized the doulbe-sides' victory of cost and efficient, on cost and efficient, all has advantage than the predictive model of present report.
Description of drawings
Fig. 1: the transporting pathway of medicine in Caco-2 cell monolayer model, wherein: (I) striden cell passive transport (cell permeability); The other transhipment of (II) cell; The transhipment (a: take in, b effluxes) that (III) is carrier mediated; (IV) transhipment after metabolism;
Fig. 2: 7 days Caco-2 cell model resistance values are along with the variation of cell cultures fate;
Fig. 3: fluorescent yellow typical curve;
Fig. 4: the see through value (mean ± variance, n=3) of fluorescent yellow in the thin model of Caco-2 individual layer;
Fig. 5: Proprasylyte typical curve;
Fig. 6: the see through value (mean ± variance, n=3) of Proprasylyte in the thin model of Caco-2 individual layer;
Fig. 7: Caco-2 cell model prediction in 7 days and external absorption correlation analysis;
Fig. 8: Caco-2 cell model prediction in 21 days and external absorption correlation analysis;
Fig. 9: digoxin typical curve;
Figure 10: the two-way transhipment characteristics of digoxin in 21 days Caco-2 cell models;
Figure 11: the two-way transhipment characteristics of digoxin in 7 days Caco-2 cell models;
Figure 12: the checking of 21 days Caco-2 cell model individual layers (hot spot is a nucleus);
Figure 13: the checking of 7 days Caco-2 cell model individual layers (hot spot is a nucleus);
Figure 14: in 21 days Caco-2 cell models owing to plant the too high multi-layer cellular (hot spot is a nucleus) that occurs of plate density.
Embodiment
The foundation of model.
Fig. 1 is the transporting pathway of Caco-2 cell model Chinese traditional medicine in cell, and the present invention sets up quick Caco-2 cell monolayer model according to these transporting pathway, i.e. said medicine oral absorption prediction and screening model, and its establishment method concrete steps are following:
(1) is basic medium with DMEM (Dulbecco ' s modified Eagle medium), contains the foetal calf serum of volume(tric)fraction 20%, 1% non-essential amino acid, 100 UmL in the basic medium
-1Penicillium mould and 100 UmL
-1Streptomycin sulphate; With the Caco-2 cell cultures in 25 cm
2In the disposable culturing bottle of cassette, place in 37 ℃ of incubators, feed 5% CO
2, cultivate under relative humidity 90% condition, change basic medium every other day;
(2) plant plate previous day with mouse tail collagen bed board.The compound method of mouse tail collagen is: gets the 100ml ultrapure water, adds 100 μ l acetate, and autoclaving, the cooling back adds 5mg/ml mouse tail collagen (giving birth to friendly technological ltd) 2ml, and with the methyl alcohol dilution, the dilution volume ratio is a methyl alcohol: mouse tail collagen=4:1 before using.On 12 hole Transwell plates, every hole adds the above-mentioned mouse tail of 800 μ L collagen, air dried overnight;
(3) when the Caco-2 cell grows to 80%～90% at the bottom of covering the culturing bottle bottle, trysinization, on 12 hole Transwell plates, inoculum density is about 5 * 10 with the Caco-2 cell inoculation
5/ hole, Transwell plate cavity of resorption adds basic medium 1.5 mL;
Change basic medium (4) the 2nd, 3 day every day, added the differentiation substratum on the 4th, 5,6 day, division culture medium makes for interpolation MIOT after adopting epithelial cell differentiation basic medium; After cultivating 6 days altogether, obtain to form the Caco-2 cell of tight individual layer.
Before the transport experiment of medicine is carried out and after finishing, all measure transepithelial cell resistance with the cell potential appearance, to confirm the compactness and the integrity of monolayer, qualified cell can be used for experiment.
Substrate or the affinity tag that adopts several kinds of standards absorbs in prediction and the screening model (below be called 7 days cell models) at the drug oral that embodiment 1 sets up and carries out transport experiment; The expression of the integrity of pair cell model, permeability, cell bypass transhipment and P-gp (P-gp) is verified; And further morphologic investigation, and experimental result and 21 days traditional Caco-2 cell models compared.
One, the integrity of cell model
The cell resistance value is to estimate the index of Caco-2 cell model integrity.We use cell potential appearance (EVOM) that the resistance value of quick Caco-2 model is measured in this research: before cell kind plate, Transwell template die resistance value is measured; Be blank value Ω 1; The resistance value of measuring when receiving plate is final resistance value Ω 2, i.e. Ω (TEER)=Ω 1-Ω 2.
Use cell potential appearance (EVOM) that the resistance value of the quick Caco-2 model of embodiment 1 foundation is measured, the result is as shown in Figure 2, and the cell resistance value is all greater than 350 Ω.Should be between 150-650 Ω, all within acceptability limit according to its resistance value of bibliographical information.
Two, cell bypass transhipment
Fluorescent yellow is to estimate the classical affinity tag that the bypass of Caco-2 cell model sees through.This experiment adopts the spectrophotofluorometer method to measure fluorescent yellow concentration.The fluorescent yellow excitation wavelength is 420nm, and emission wavelength is 520nm.
20ng/mL fluorescent yellow is added on Caco-2 cell monolayer top (A face), behind 3h, detects from base terminal (B face) sampling.
The apparent PQ of medicine (Papp) is according to computes:
Papp=[(dC/dt(V)]/(A×C
0)
C
0Be the medicine starting point concentration of dosing side, dC/dt is for accepting the speed that the side medicine occurs, and V is a liquor capacity of accepting side, and A is the surface-area of Transwell poly carbonic ether film.
The typical curve equation of fluorescent yellow is in HBSS as shown in Figure 3 (Hanks Balanced Salt Solutions, the hank's balanced salt solution) solution: Y=157.4X-26.46, r=0.999 (n=5).Linearity range is 31.25～2000ng/ml.Wherein Y is the absorbance (μ g/ml) of fluorescent yellow, and X is fluorescent yellow concentration (μ g/ML).The compactness of visible Caco-2 cell monolayer as shown in Figure 4 is good, and fluorescent yellow was at 21 days cell models and distinguished 0.176 and 0.159 (10 through value in the cell model in 7 days
-6Cm/s), no difference of science of statistics as a result, 7 days cell models maybe be because incubation time reduces, and uncertain factor reduces action time, and variance is less.
Three, cell permeability
Proprasylyte is a classical affinity tag of estimating Caco-2 cell permeability.The HPLC method of Proprasylyte in the detection of biological sample is at first set up in this experiment.The employing chromatographic column is HypersilBDSC18 (I.D.4.6mm * 150mm, 5 a μ m) stainless steel column (Dalian produces according to Lyntech Corporation (US) 10177 South 77th East Avenue Tulsa, Oklahoma 74133 U.S.); Moving phase is the 25mM potassium phosphate buffer: acetonitrile (60:40), transfer pH to 2.5 with phosphoric acid; Flow velocity 1.0ml/min; Detect wavelength 290nm; Sample size 20 μ L; Adopt verapamil 150 μ g/mL as interior mark.100 μ mol/L fluorescent yellows are added on Caco-2 cell monolayer top (A face), behind 1h, detect from base terminal (B face) sampling.
The typical curve equation of Proprasylyte is in the HBSS solution as shown in Figure 5: Y=0.098X-0.246, r=0.995 (n=5).Linearity range is 1～200 μ mol/L.The permeability of result's visible Caco-2 cell monolayer as shown in Figure 6 is good, and Proprasylyte was at 21 days cell models and be respectively 31.36 and 32.44 (10 through value in the cell model in 7 days
-6Cm/s), no difference of science of statistics is consistent through value with fluorescent yellow as a result, and it is less that Proprasylyte sees through the value variance in 7 days cell models.
Four, the correlation analysis of human body oral absorption availability and the medicine value of seeing through in cell
In the Caco-2 cell model, see through the result according to medicine, in conjunction with reporting that medicine at human body oral absorption availability, carries out correlation analysis.
By correlation analysis Fig. 7, shown in Figure 8, Proprasylyte and the fluorescent yellow value of seeing through and the human body oral absorption availability dependency in the Caco-2 cell monolayer is good.And 7 days cell models and 21 days cell models predict the outcome almost consistent, shown in result such as the table 1, table 2.
Table 1: fluorescent yellow inside and outside correlation analysis
Table 2: Proprasylyte inside and outside correlation analysis
Five, the transhipment of classical P gp (P-gp) substrate digoxin in the Caco-2 model
P gp (P-gp) is one of most important translocator in the human body; Relate to medicine in absorption of human body; Various procedures in distribution and the metabolism; Now because many antitumor drugs are substrates of P-gp and can't reach effective Plasma Concentration, cause therapeutic action not good (like taxol etc.), confirm at the initial stage of medicament research and development whether medicine is that the P-gp substrate has important practical significance to instructing compound structure transformation, clinical application etc.
According to the governing principle of FDA, if compound is that the 2-3 of A-B can think that medicine is the substrate of P-gp more than doubly in the transhipment amount of Caco-2 model Chinese traditional medicine B-A.Digoxin is the classical substrate of P-gp, and the transhipment amount of its B-A of bibliographical information is 4-26 a times of A-B.
The HPLC-MS/MS method of digoxin in the detection of biological sample is at first set up in this experiment: after the sample ammonification scaleization and adopt extraction agent (t-butyl methyl ether: methylene dichloride=3:1) carries out liquid-liquid extraction.Ion source is electro-spray ionization source (ESI); Detection mode is that positive ion detects; Scan mode adopts selective reaction monitoring (SRM).The stratographic analysis post be C18 XTerra MS analytical column (3.5 μ m, 2.1 * 50 mm, Waters, USA); Moving phase be methanol-water (containing 5 mM ammonium formiates+0.1% formic acid) (80:20, v/v); Flow velocity is 200 μ L/min; Be 2 min analysis time.0.5 μ mol/L digoxin is added on back, Caco-2 cell monolayer top (A face) to be detected from base terminal (B face) sampling respectively at 30min, 60min, 80min, 100min, 120min.Simultaneously 0.5 μ mol/L digoxin being added on back, Caco-2 cell monolayer top (B face) detects from base terminal (A face) sampling respectively at 30min, 60min, 80min, 100min, 120min.
As shown in Figure 9, the typical curve equation of digoxin is in the HBSS solution: Y=0.034X-0.059 R
2=0.998 (n=5), linearity range is 0.5～100 μ mol/L.The ratio of positive drug digoxin B-A and A-B is 6.2 in visible 7 days cell models in table 3; According to high B-A in bibliographical information ground and A-B ratio range is 4-26; Can know that 7 days cell model ratio is in the scope of report; But cell model ratio was less in relative 21 days, possibly be because P-gp expression amount relative deficiency.Result such as Figure 10 and shown in Figure 11, cell model also can reflect the transhipment characteristics through P-gp mediation medicine intuitively in 7 days, cell model still was applicable to the transport of drug experiment of carrying out the P-gp mediation in visible 7 days.
Table 3: the characteristics of Gaoxin two-way transhipment in 21 days cell models and 7 days cell models comparatively
Six, the morphologic investigation of Caco-2 monolayer
21 days Caco-2 cell models of tradition relatively, the density of 7 days Caco-2 cell model when kind of plate is higher, because cell monolayer all possibly impact with transhipment the homogeneity of model, therefore many reports are all pointed out the importance of cell monolayer.This experiment, is handled with 1% Triton pair cell Caco-2 monolayer rinse 3 times with PBS, and each 5min repeats 3 times.Use DAPI (1 μ g/ml) under the lucifuge condition, to dye again, clean background dyeing with PBS behind the 12h.(Axiovert, ZeissCLSM 501, Jena, Germany) observation down as for Laser Scanning Confocal Microscope with staining cell.The excitation wavelength of DAPI is 385 nm.Handle with Zeiss LSM 510 software packages behind the IMAQ.
Be the cell monolayer model like Figure 12,21 days shown in Figure 13 and 7 days models, the situation of the multi-layer cellular of bibliographical information (Figure 14) do not occur.
Claims (5)
1. a drug oral absorbs the establishment method of prediction and screening model, it is characterized in that comprising the steps:
(1) is base culture base Caco-2 cell with DMEM, contains the foetal calf serum of volume(tric)fraction 20%, 1% non-essential amino acid, 100 UmL in the basic medium
-1Penicillium mould and 100 UmL
-1Streptomycin sulphate;
(2) the kind plate promptly adds mouse tail collagen, air dried overnight with mouse tail collagen bed board previous day in each hole on the Transwell plate; The compound method of mouse tail collagen is: gets the 100ml ultrapure water, adds 100 μ l acetate, and autoclaving, the cooling back adds 5mg/ml mouse tail collagen 2ml, and with the methyl alcohol dilution, the dilution volume ratio is a methyl alcohol: mouse tail collagen=4:1 before using;
(3) when the Caco-2 cell grows to 80%～90% at the bottom of covering the culturing bottle bottle, trysinization, on the Transwell plate, Transwell plate cavity of resorption adds basic medium with the Caco-2 cell inoculation;
Change basic medium (4) the 2nd, 3 day every day, added the differentiation substratum on the 4th, 5,6 day, division culture medium makes for interpolation MIOT after adopting epithelial cell differentiation basic medium; After cultivating 6 days altogether, obtain to form the Caco-2 cell of tight individual layer.
2. drug oral as claimed in claim 1 absorbs the establishment method of prediction and screening model, it is characterized in that cultivating the Caco-2 cell in the step (1) is that the Caco-2 cell is put into culturing bottle, places in the incubator and cultivates, and changes basic medium every other day; The culture condition of incubator is: 37 ℃ of temperature feed 5% CO
2, relative humidity 90%.
3. drug oral as claimed in claim 1 absorbs the establishment method of prediction and screening model, it is characterized in that adding in each hole in the step (2) mouse tail collagen 800 μ L.
4. drug oral as claimed in claim 1 absorbs the establishment method of prediction and screening model, and the inoculum density that it is characterized in that step (3) is 5 * 10
5/ hole.
5. drug oral as claimed in claim 1 absorbs the establishment method of prediction and screening model, it is characterized in that step (3) Transwell plate cavity of resorption adds basic medium 1.5 mL.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104357398A (en) * | 2014-11-07 | 2015-02-18 | 四川省中医药科学院 | Commissural cell model as well as preparation method and application thereof |
| CN107083364A (en) * | 2017-01-13 | 2017-08-22 | 江苏省农业科学院 | A kind of structure of Caco-2/HUVEC cells co-culture system |
| CN109321529A (en) * | 2018-10-19 | 2019-02-12 | 浙江工商大学 | A kind of construction method of external gastrointestinal model and application |
-
2012
- 2012-02-02 CN CN2012100231420A patent/CN102533927A/en active Pending
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| 《Journal of Pharmaceutical Sciences》 20020331 Shinji Yamashita，et al "New and better protocols for a short-term Caco-2 cell culture system" 669-679 1-5 第91卷, 第3期 * |
| 《中国实验方剂学杂志》 20110630 马博等 "肠上皮细胞模型不同培养条件的优化及适应性研究" 第205-210页 1-5 第17卷, 第11期 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104357398A (en) * | 2014-11-07 | 2015-02-18 | 四川省中医药科学院 | Commissural cell model as well as preparation method and application thereof |
| CN107083364A (en) * | 2017-01-13 | 2017-08-22 | 江苏省农业科学院 | A kind of structure of Caco-2/HUVEC cells co-culture system |
| CN109321529A (en) * | 2018-10-19 | 2019-02-12 | 浙江工商大学 | A kind of construction method of external gastrointestinal model and application |
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Application publication date: 20120704 |