A new method for evaluating the intestinal permeability of drugs

Abstract

The present invention provides a new method for evaluating the intestinal permeability of drugs. Small intestinal organoid cells and Caco-2 cells are co-cultured in the #imgabs0# device to form a dense cell layer to simulate the intestinal wall, and the intestinal wall permeability test of the drug is completed within the #imgabs1# device, and the apparent permeability coefficient, efflux ratio and recovery rate of the drug to be tested are calculated; the small intestinal organoid cells are obtained by reprogramming and directed differentiation of PBMC cells. By mixing the induced differentiated small intestinal organoid cells with the commonly used colon cancer cell line Caco-2 in different proportions, the present invention finds that the mixed cells can not only form a complete and dense cell layer faster, and its resistance can reach ≥500 Ω / cm 2 or more about 7 days after plating, and the dense cell layer formed thereby can be effectively used to evaluate the intestinal permeability of compounds in vitro, just like the Caco-2 cell layer cultured for 20 days.

Description

Technical Field

[0001] The present invention relates to a new method for evaluating the intestinal permeability of drugs. Background Art

[0002] The most common administration route for small molecule drugs is oral administration. Many candidate drug molecules have very low bioavailability during preclinical development because they cannot be effectively absorbed through the intestine, resulting in drug development failure. Therefore, it is necessary to conduct detailed research and identification on the intestinal permeability of candidate drug molecules. Preclinical research methods are divided into in vitro permeation models and in vivo PK experiments in animals. Among them, the commonly used in vitro permeation models are Caco-2 permeation experiments and MDCK permeation experiments. In vivo PK experiments in animals refer to oral administration and intravenous administration in experimental animals such as mice, rats, dogs or monkeys respectively, so as to calculate the bioavailability and evaluate the intestinal permeation ability of the compound.

[0003] In order to simulate the absorption process of compounds in the small intestine, the Caco-2 cell permeation method has been popular since the 1980s. Caco-2 cells are a type of human intestinal cancer cells. During the experiment, these cells are inoculated in the upper culture wells of a special cell culture device of clusters. The bottom of the upper culture wells is a semi-permeable membrane, and then this is stacked on the corresponding lower culture wells, and the semi-permeable membrane is immersed in the culture medium in the lower culture wells. After inoculation, the culture medium is changed once a day. After about 21 days of culture, the transepithelial electrical resistance (TEER) of the cell layer is measured to evaluate the integrity and tightness of the cell layer. In this device, as the cell density increases, Caco-2 cells change from flat epithelial cells to columnar epithelial cells and form tight junctions between cells. At the same time, Caco-2 cells express various enzymes in normal human small intestinal epithelial cells, such as aminopeptidase, alkaline phosphatase, etc. Therefore, this model is often used to evaluate the intestinal permeation ability of drugs.

[0004] After the TEER reaches the required value, it indicates that the Caco-2 cells have formed a dense cell layer, and then the experiment can be carried out. The apical membrane (denoted as layer A) of the intestinal cells corresponding to the upper middle layer, and the basement membrane (denoted as layer B) of the intestinal cells corresponding to the lower part. A compound with a specific concentration is added to the upper wells or the lower culture wells. After culturing for a certain period of time, the content of the compound in the lower or upper culture medium is measured, and then the permeability parameter of the compound can be calculated.

[0005] In the current technology, the first problem is that the culturing time is long. Usually, at least 21 days of continuous culturing is required for the TEER of Caco-2 to reach the target value, which usually needs to be greater than 1900 Ω. Secondly, although Caco-2 cells are easy to grow and form a polarized monolayer, since the cell types in the formed monolayer cell membrane are single, there are no goblet cells, and there are also lacks of intercellular junctions formed between different types of cells, the data obtained through this model sometimes cannot accurately predict the permeability coefficient of candidate drugs. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the defects existing in the prior art. The present invention proposes a new method for evaluating the intestinal permeability of drugs to solve the problems mentioned in the above background art.

[0007] To solve the above technical problems, the technical solution adopted by the present invention is as follows: A new method for evaluating the intestinal permeability of drugs, and the specific process is as follows: Co-culture small intestinal organoid cells and Caco-2 cells to form a dense cell layer, and the dense cell layer can be used to simulate the intestinal wall. Within the device, complete the drug intestinal wall permeability test and calculate the apparent permeability coefficient, efflux ratio, and recovery rate of the drug to be tested.

[0008] The small intestinal organoid cells are obtained by reprogramming and directed differentiation of PBMC cells;

[0009] Co-culture the small intestinal organoid cells and Caco-2 cells to obtain a dense cell layer, and the cell layer resistance is not less than 500 ohms / cm 2 , for a 24-well its the semi-permeable membrane area in the well is about 0.33 cm 2 , and the resistance of the blank control well is usually about 150 ohms. Therefore, its total resistance should generally not be less than 1900 ohms;

[0010] During the drug permeability test, the intestinal surface is set on the upper layer of the device, and the basal layer is set on the lower layer of the device. The specific process of the drug intestinal permeability test is as follows: Set an HBSS solution containing the drug to be tested at the donor end, and set a blank HBSS solution at the receiver end for the permeability test. The donor end can be either the intestinal surface or the basal layer, and the receiver end is the other one of the intestinal surface or the basal layer.

[0011] Furthermore, after the device is incubated in a 37°C, 5% CO2 cell culture incubator for a certain period of time, equal amounts of solution are taken from the donor end and the receiver end, and the concentration of the drug to be tested in them is measured.

[0012] Furthermore, the apparent permeability coefficient: Papp = C Rec × V Rec / (A × t × C0),

[0013] where C Rec is the concentration of the sample at the receiver side; V Rec is the volume of the sample at the receiver side; A is the surface area of the Transwell membrane; t is the incubation time (seconds); C0: the initial drug concentration at the donor side;

[0014] Efflux ratio: Efflux Ratio = Papp(B→A) / Papp(A→B),

[0015] where Papp(B→A) is the permeability parameter from the basal layer to the intestinal surface, and Papp(A→B) is the permeability parameter from the intestinal surface to the basal layer;

[0016] Recovery rate = [(V Rec × C Rec ) + (V d × C d )] / (V d × C0),

[0017] where V d and V Rec are the volumes of the solution at the donor side and the receiver side, respectively; C Rec and C d are the drug concentrations at the receiver side and the donor side at the time of sampling, respectively.

[0018] Furthermore, during the co-culture process, after mixing the small intestinal organoid cells and Caco-2 cells, they are resuspended in the culture medium and then inoculated into the device; the number of cells inoculated in each well of the 24-well plate is 3X10 to 6X10 4 to 6X10 4 ; among them, the proportion of small intestinal organoid cells is 20-70%, and the proportion of Caco-2 cells is 30-80%.

[0019] Furthermore, the small intestinal organoid cells are obtained by the directional differentiation of induced pluripotent stem cells (iPSCs) using a kit, and an identification process of small intestinal organoid cells is included. During the identification process, it includes: FOXA2 immunofluorescence staining, CDX2 immunofluorescence staining, flow cytometry analysis of the expression of EpCAM and LGR5, and detection of the expression of the FCGRT protein by immunoblotting.

[0020] Furthermore, the small intestine organoid cells are induced pluripotent stem cells (iPSCs) reprogrammed from PBMC cells using a reprogramming kit; an iPSC verification process is also included. During the verification process, teratoma formation assay and TRA-1-60 immunofluorescence staining are used to confirm that the reprogrammed induced pluripotent hepatocytes possess pluripotency.

[0021] Compared with the prior art, the beneficial effects of the present invention include: by mixing induced differentiated small intestine organoid cells with the commonly used colon cancer cell line Caco-2 in different ratios, it is found that the mixed cells can not only form a complete and dense cell layer faster, and the electrical resistance can reach ≥500 Ω / cm 2 or more about 7 days after plating, and the dense cell layer formed thereby can be effectively used to evaluate the intestinal permeability of compounds in vitro, just like the Caco-2 cell layer cultured for 20 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The disclosure of the present invention will be described with reference to the accompanying drawings. It should be understood that the drawings are only for illustrative purposes and are not intended to limit the scope of protection of the present invention. In the drawings, the same reference numerals are used to refer to the same components. Among them:

[0023] Figure 1 Schematically shows the curve of the electrical resistance of the co-culture cell layer of cells with different ratios changing with time;

[0024] Figure 2 Schematically shows the curve of the electrical resistance of cell layers with different seeding densities changing with time;

[0025] Figure 3 Schematically shows the microscopic observation results (40X) of the co-culture of 40% small intestine organoids and Caco-2 cells in Transwell for 5 days. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0026] It is easy to understand that according to the technical solution of the present invention, without changing the essence of the present invention, those of ordinary skill in the art can propose various structural and implementation methods that can be mutually replaced. Therefore, the following detailed description and the accompanying drawings are only illustrative of the technical solution of the present invention and should not be regarded as the whole of the present invention or as a limitation or restriction on the technical solution of the present invention.

[0027] A new method for evaluating the intestinal permeability of drugs includes the following steps:

[0028] S1. Reprogram PBMC cells into induced pluripotent stem cells (iPSCs) using a somatic cell reprogramming kit:

[0029] In this application, PBMC cells from healthy volunteers were first isolated and reprogrammed into induced pluripotent stem cells (iPSCs) using a commercial somatic cell reprogramming kit (such as the ReproRNA TM OKSGM or Thermo CytoTune TM -iPS 2.0 Sendai virus reprogramming kit) of STEMCELL Technologies Inc. The reprogrammed iPSCs were confirmed to have pluripotency through tests such as teratoma formation.

[0030] S2. Differentiate the iPSCs into intestinal organoids by directed differentiation:

[0031] Use the STEMdiff TM Intestinal OrganoidKit kit from Stem Cell Technologies Inc. to reprogram the iPSCs into intestinal organoids according to the operation manual provided by the manufacturer. Then, identify the differentiated intestinal organoids, including: FOXA2 immunofluorescence staining, CDX2 immunofluorescence staining, flow cytometry analysis of the expression of EpCAM and LGR5, and detection of the expression of FCGRT protein by immunoblotting.

[0032] S3. Co-culture the intestinal organoid cells with Caco-2 cells in the device:

[0033] Take intestinal organoid (IO) cells and Caco-2 cells, mix them in different proportions. The proportion of intestinal organoid cells ranges from 0 - 90%, and the proportion of Caco-2 cells ranges from 10% - 100%. After mixing the cells, take a total of 30,000 - 60,000 cells, resuspend them in 300 μL of culture medium, and add them to the upper wells of a 24-well Transwell. Add 1 mL of culture medium to the lower wells. For other specifications of Transwell devices or similar devices, the number of cells inoculated may be different. After inoculation, change the culture medium once a day.

[0034] S4. Detect the tightness of the co-cultured cell layer by measuring the transepithelial electrical resistance of the cell layer:

[0035] The transepithelial electrical resistance of the cell layer in can be monitored using an EVOM2 transepithelial electrical resistance meter (TEER) from World Precision Instruments or a similar instrument to evaluate the tightness and integrity of the cell layer. Before measurement, soak the measurement electrodes in 75% isopropyl alcohol for 15 minutes for disinfection, and then soak them in PBS. First, measure the Add HBSS buffer to the small holes and the lower holes, then measure the resistance between the upper and lower layers, which is the blank resistance. Then measure the resistance between the upper and lower layers in the wells inoculated with cells, denoted as the total resistance; then subtract the blank resistance from the total resistance, which is the cellular resistance of this well. Generally, for a 24-well device, its 2 area is 0.33 cm

[0036] S5. In vitro intestinal wall permeability test of drugs:

[0037] Prepare a 10 μM or other concentration HBSS working solution of the control compound and the drug to be tested, and then wash the wells with cells paved with HBSS three times. Upper wells and lower wells. The apical membrane (denoted as A) of the intestinal cells corresponding to the upper surface of the cell layer, and the basement membrane (denoted as B) of the intestinal cells corresponding to the lower surface. For the intestinal permeability test of each compound, the permeability from A to B (A→B) and the permeability from B to A (B→A) need to be measured respectively. Therefore, generally for the intestinal permeability test of each compound, 4 wells are required. For the A→B permeability test, two replicate wells, in the upper wells (donor ends), add 200 μL of the HBSS working solution containing the compound respectively, and add 700 μL of the blank HBSS solution without the compound in the lower wells (receiver ends); for the B→A permeability test, two replicate wells, in the upper wells (receiver ends), add 200 μL of the blank HBSS solution without the compound respectively, and add 700 μL of the HBSS working solution containing the compound in the lower wells; after adding the drugs, place the entire microplate back into the cell culture incubator at 37 °C and 5% CO2 for incubation for a certain period of time. After the incubation is over, take 50 μL of the solution from the upper wells and the lower wells respectively, and determine the concentration of the compound in them by liquid chromatography-mass spectrometry.

[0038] S6. Calculate the permeability parameters of the compound:

[0039] Calculate the apparent permeability coefficient Papp of the compound to be measured according to the formula Papp = C Rec ×V Rec / (A × t × C0). Where:

[0040] C Rec is the concentration of the sample at the receiver end; V Rec is the volume of the sample at the receiver end; A is Membrane surface area; t is the incubation time (seconds); C0: initial drug concentration at the donor end;

[0041] Efflux Ratio: Efflux Ratio = Papp(B→A) / Papp(A→B),

[0042] wherein, Papp(B→A) is the permeability parameter from the basal layer to the intestinal surface, and Papp(A→B) is the permeability parameter from the intestinal surface to the basal layer;

[0043] Recovery rate = [(V Rec ×C Rec )+(V d ×C d )] / (V d ×C0),

[0044] wherein, V d and V Rec are the solution volumes at the donor end and the receiver end respectively; C Rec and C d are the drug concentrations at the receiver end and the donor end at the time of sampling respectively.

[0045] The following specifically describes the technical effects of a new method for evaluating the intestinal permeability of drugs in the present invention in conjunction with examples.

[0046] Example 1: Changes in resistance in the cell dense layer formed by mixing small intestinal organoid cells and Caco-2 cells in different proportions Take the identified small intestinal organoid cells and Caco-2 cells, and mix them according to the proportions in Table 1 below.

[0047] Table 1 Numbers of intestinal organoids and Caco-2 cells in different proportion combinations

[0048] Number Small intestinal organoid cells Caco-2 cells R1 0 100％ R2 10％ 90％ R3 20％ 80％ R4 30％ 70％ R5 40％ 60％ R6 50％ 50％ R7 60％ 40％ R8 70％ 30％ R9 80％ 20％ R10 90％ 10％

[0049] After mixing cells in different proportions, take a total of 50,000 cells, resuspend them in 300 μL of culture medium, and add them to the upper wells of 24-well Corning Add 1 mL of culture medium to the lower wells. Each number has 2 replicate wells. After cell seeding, start measuring the resistance between the upper and lower layers in each well using an EVOM2 epithelial resistance measuring instrument the next day (recorded as the first day). The resistance results of each number every day are shown in Table 2 below: Add 1 mL of culture medium to the lower wells. Each number has 2 replicate wells. After cell seeding, start measuring the resistance between the upper and lower layers in each well using an EVOM2 epithelial resistance measuring instrument the next day (recorded as the first day). The resistance results of each number every day are shown in Table 2 below:

[0050] Table 2 Epithelial resistance data of cell combinations with different numbers

[0051]

[0052] As the culture time extended, the small intestinal organoid cells in wells R2 - R8 formed cell aggregates and were prone to detachment when moving the microwell plate, thus affecting the compactness and integrity of the cell layer. Therefore, their resistance was not measured in the later stage of the experiment. The resistance of each numbered sample was plotted against the number of incubation days, and the results are shown in Figure 1 . The results showed that Caco-2 cells (R1) without mixed small intestinal organoid cells required 20 days to form a compact cell layer, which was consistent with the method reported in the literature. When 10% - 70% of small intestinal organoid cells were mixed with Caco-2 cells, a compact cell layer could be formed faster, that is, the measured resistance was greater than 1900 ohms. In particular, for R5, R6, and R7 (the corresponding proportions of small intestinal organoid cells were 40%, 50%, and 60% respectively), a compact cell layer could be formed on the 7th day of culture, that is the transepithelial electrical resistance across the upper and lower layers was stably greater than 1900 ohms. However, when the proportion of small intestinal organoid cells was increased to more than 80%, the resistance of the formed cell layer decreased, indicating that the required compact cell layer could not be formed. The results showed that mixing an appropriate amount of small intestinal organoid cells with Caco-2 could significantly increase the speed of forming tight junctions between cells and significantly shorten the time to form a compact cell layer.

[0053] Example 2: Effect of cell seeding density on the formation of cell compact layer

[0054] To investigate the effect of cell seeding density on the formation of a complete and compact cell layer, identified small intestinal organoid cells and Caco-2 cells were mixed at a ratio of 50%:50%. A total of 3×10 4 , 4×10 4 , 5×10 4 and 6×10 4 cells were taken, resuspended in 300 μL of culture medium, and added to the upper wells of 24-well Corning . 1 mL of culture medium was added to the lower wells of . There were 2 replicates for each condition. After cell seeding, the transepithelial electrical resistance between the upper and lower layers of each well was measured using an EVOM2 transepithelial resistance meter the next day (the first day). The daily resistance measurement results are shown in Table 3.

[0055] Table 3 Cell layer resistance under different seeding density conditions (unit: ohm)

[0056]

[0057]

[0058] The resistance at different seeding densities was plotted against the number of incubation days, and the results are shown in Figure 2The results showed that the formation speed of the dense cell layer was related to the cell seeding density, and the seeding number was 3x10 4 , 4x10 4 , 5x10 4 With 6x10 4 It takes about 12 days, 9 days, 7 days, and 7 days for the mixed cells to form a dense cell layer, that is, the measured resistance is >1900 ohms. The results show that in Corning 5x10 cells were seeded in the upper wells of the 24-well plate. 4 or 6x10 4 The 1:1 mixture of small intestinal organoid cells and Caco-2 cells can form a dense cell layer in about 7 days. A decrease in the number of cells will prolong the time required to form a dense cell layer.

[0059] Example 3: Testing the permeability of control compounds using a dense layer of cells formed by mixing small intestinal organoid cells and Caco-2 cells in a preferred ratio

[0060] In order to test the permeability of the cell layer formed by small intestinal organoid cells and Caco-2 cells, we selected two control compounds, digoxin and propranolol, for evaluation. Digoxin is a substrate of pgp and is easily excreted by intestinal cells, so it is a compound with medium and low permeability; propranolol has good intestinal permeability in the human body and is a high permeability compound. We chose to test the cell permeability in three ratios, R5, R6, and R7, and compared them with R1, which is a cell layer formed by only Caco-2. Each ratio combination requires 12 wells, 6 wells for digoxin and propranolol each, and then 3 wells to test the permeability from end A to end B, and 3 wells to test the permeability from end B to end A. Prepare cell combinations of different ratios, namely R5, R6, R7 and R1, according to the above method, and lay 12 wells in each 50,000 cells were plated in each well. Then, the cell resistance of R5, R6 and R7 cell combinations was tested on the 7th day. The resistance across the cell layer was >1900 ohms. 10 μM digoxin and propranolol HBSS solutions were added to the wells according to the above method. The upper or lower wells were used to test the permeability of A→B and B→A. For the R1 combination, the cell resistance was tested on day 21, and the cell resistance was >1900 ohms. Then the permeability of digoxin and propranolol was tested in the same way.

[0061] The results are shown in Table 4. One-way ANOVA multiple comparison analysis was performed on the permeability of digoxin and propranolol in the tested numbered cell combinations. There were no significant differences among the groups in terms of either the recovery rate, the apparent permeability coefficient (Papp), or the efflux rate (p > 0.05). This indicates that both the R5, R6, and R7 groups can be used to evaluate the intestinal wall cell permeability of compounds in vitro in the same way as the R1 group. That is, the dense cell layer formed by mixing 40%-60% of small intestinal organoid cells with Caco-2 cells can not only form a complete dense cell layer faster, but also the data measured from it is consistent with that of the dense cell layer composed of only Caco-2 cells, and can be used for the evaluation of the intestinal wall permeability of compounds in vitro.

[0062] Table 4 Permeability of digoxin and propranolol in different cell combinations

[0063]

[0064]

[0065] The technical scope of the present invention is not limited to the content described above. Those skilled in the art can make various deformations and modifications to the above embodiments without departing from the technical idea of the present invention, and these deformations and modifications should all fall within the protection scope of the present invention.

Claims

1. A new method for evaluating the intestinal permeability of drugs, characterized in that, The specific process is as follows: Small intestine organoid cells and Caco-2 cells are co-cultured in a Transwell® device to form a dense cell layer, which can be used to simulate the intestinal wall of the small intestine. The drug intestinal wall permeability test is carried out within the Transwell® device, and the apparent permeability coefficient, efflux ratio, and recovery rate of the drug to be tested are calculated. The small intestine organoid cells are obtained by reprogramming and directed differentiation of PBMC cells. Co-culture the small intestine organoid cells and Caco-2 cells to obtain a cell layer, and the resistance of the cell layer is not less than 500 Ω / cm 2 ; The intestinal surface is set on the upper layer of the Transwell® device, and the basal layer is set on the lower layer of the Transwell® device. The specific process of the drug intestinal permeability test is as follows: An HBSS solution containing the drug to be tested is set at the donor end, and a blank HBSS solution is set at the receiver end for the permeability test. The donor end can be either the intestinal surface or the basal layer, and the receiver end is the other one of the intestinal surface or the basal layer.

2. The new method for evaluating the intestinal permeability of a drug according to claim 1, characterized in that, After the Transwell® device is incubated in a 37°C, 5% CO2 cell culture incubator for 90 minutes, equal amounts of solution are taken from the donor end and the receiver end, and the concentration of the drug to be tested in them is measured.

3. The new method for evaluating the intestinal permeability of a drug according to claim 2, characterized in that, The apparent permeability coefficient: Papp = C Rec ×V Rec / (A × t × C0); Among them, C Rec is the concentration of the sample at the receiving end; V Rec is the volume of the sample at the receiving end; A is the surface area of the Transwell membrane; t is the incubation time; C0 is the initial drug concentration at the donor end; Efflux Ratio: Efflux Ratio = Papp(B→A) / Papp(A→B); Among them, Papp(B→A) is the apparent permeability parameter from the basal layer to the intestinal surface, and Papp(A→B) is the apparent permeability parameter from the intestinal surface to the basal layer. Recovery rate = [(V Rec × C Rec ) + (V d × C d )] / (V d × C0); Among them, V d and V Rec are the solution volumes at the supply end and the receiving end respectively; C Rec and C d are the drug concentrations at the receiving end and the supply end during sampling respectively.

4. The new method for evaluating the intestinal permeability of a drug according to claim 1, characterized in that, During the co-culture process, after mixing intestinal organoid cells with Caco-2 cells, they are resuspended in the culture medium and then inoculated into a Transwell® device; the number of cells inoculated in each Transwell® in a 24-well plate is 3×10 4 to 6×10 4 ; among them, the proportion of intestinal organoid cells is 20-70%, and the proportion of Caco-2 cells is 30-80%.

5. The new method for evaluating the intestinal permeability of drugs according to claim 1, characterized in that, The small intestine organoid cells are obtained by directed differentiation of induced pluripotent stem cells (iPSCs), and the identification process of the small intestine organoids is included. The identification process includes: FOXA2 immunofluorescence staining, CDX2 immunofluorescence staining, flow cytometry analysis of the expression of EpCAM and LGR5, and detection of the expression of the FCGRT protein by immunoblotting.

6. The new method for evaluating the intestinal permeability of a drug according to claim 5, characterized in that, The small intestine organoid cells are induced pluripotent stem cells (iPSCs) reprogrammed from PBMC cells, and the verification process of the induced pluripotent stem cells is included. During the verification process, the teratoma formation test and TRA-1-60 immunofluorescence staining are used to confirm whether the reprogrammed induced pluripotent stem cells have pluripotency.

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