Caco-2 Cell Monolayer Permeability Experiments

Assay description

Caco-2 cell monolayer permeability experiments

The Caco-2 cell mono-layers are washed once with HBSS prior to start. TEER is measured both before and after performing all the transport experiments. Experiments are made in apical A to basolateral B direction. Transport buffer, 800 µL, (HBSS, pH 7.4) is first dispensed to the basal side of the monolayer. The assay is then initiated when 200 µL of each compound is added to the apical side at 10 µM (all test compounds are diluted in HBSS, pH 6.5, with 1% DMSO as co-solvent). Samples are withdrawn before and at 45 and 120 min post addition of test compound. Two µL and 200 µL are withdrawn from the apical donor compartment and the basolateral receiver compartment, respectively. During incubation the transwell plates are placed in a shaking incubator at 480 rpm and 37°C between the sampling up to 120 min. All samples are analysed subsequently (never stored or frozen).

Caco-2 cell bidirectional transport

The Caco-2 cell monolayers are washed once with HBSS prior to start. TEER is measured both before and after performing all the transport experiments. Experiments are made in apical (A) to basolateral (B) direction and B to A direction in singlicates. The robot executes a script that runs the compounds first in AB direction and then in BA direction in sequence.

For AB direction: transport buffer, 800 μL, (HBSS, pH 7.4) is first dispensed to the basal side of the monolayer. The assay is then initiated when 200 μL of compound solution is added to the apical side at 10 or 1 μM (all test compounds are diluted from DMSO stock plates in HBSS, pH 7.4, with a final DMSO concentration of 1%). Donor samples are withdrawn at 0 and 60 minutes post addition of test compound and receiver samples are withdrawn at 60 minutes. Two μL and 200 μL are withdrawn from the apical (donor) compartment and the basolateral (receiver) compartment, respectively. During incubation between sampling the transwell plates are placed in a shaking incubator at 480 rpm and 37°C, up to the last sampling time at 60 minutes.

For BA direction: transport buffer, 200 μL, (HBSS, pH 7.4) is first dispensed to the apical side of the monolayer. The assay is then initiated when 800 μL of compound solution is added to the basal side at 10 or 1μM (all test compounds are diluted from DMSO stock plates in HBSS, pH 7.4, with a final DMSO concentration of 1%). Donor samples are withdrawn at 0 and 30 minutes post addition of test compound and receiver samples are withdrawn at 30 minutes. One and 100 μL are withdrawn from the basal (donor) compartment and the apical (receiver) compartment, respectively. During incubation between the sampling the transwell plates are placed in a shaking incubator at 480 rpm and 37°C, up to the last sampling time at 30 minutes.

Detailed Descriptions of assay condtions

Clint human hepatocytes

Cryopreserved hepatocytes are purchased from commercial sources. Percoll solution is prepared when needed and needs to be at room temperature at the time of use. Cryopreserved cells are moved from –150°C freezer and immediately immersed in a preheated water bath kept at 37°C. When only a small ice crystal can be seen the content is emptied into hepatocyte incubation media and centrifuged. In some cases when viability is low an additional step with percoll needs to be performed. The supernatant is removed and the resulting pellet is resuspended in hepatocyte incubation media. The cells are counted using Casy cell counter. An acceptable viability should be at least 75% and preferably over 80%. The cells are diluted to a concentration of 2 million cells/ml in hepatocyte media and should be used as soon as possible.

10 mM stock solutions of the test compounds are prepared in DMSO and 1 µl of each solution is dispensed into plates by the Compound Management Team (CMT). One CMT plate is ordered for each set of compounds designated for testing in hepatocytes from a particular species. The positions of the compounds in each plate must not overlap with each other. Example of further dilution when running two species on the same plate looks like this. The plates are received from CMT and 99 µl of ACN/dH2O (1:1) are added to each well, giving a compound concentration of 100 µM. 75 µl are aspirated from the CMT plate for species 1, followed by aspiration of 75 µl from the CMT plate for species 2. The 150 µl are dispensed to an empty plate, which results in a compound concentration of 50 µM. 192 µl hepatocyte medium are aspirated, followed by aspiration of 8 µl from the 50 µM solution. The 200 µl are dispensed to an empty plate, resulting in a final compound concentration of 2 µM.

The final solvent concentrations in the incubations will be 0.01 % DMSO and 1 % ACN, except for the blanks, which will have no DMSO and no ACN.

Hepatocyte suspension (2 million cells/ml) is manually added to 96-well plates (25 µl/well. Cells are also added to the blanks, which are placed on a separate plate.

Each plate represents a single time point e.g. 2, 15, 30, 45 or 60 min. The plates are pre-incubated for 10-23 min at 37°C along with the 2 µM substrate plate. Reactions are started by adding 25 µl of 2 µM substrate to the cells, starting with the longest time point, giving a final substrate concentration of 1 µM and a cell concentration of 1 million cells/ml. The Biomek FXP is used for this step. Directly after compound addition the plate is automatically shaken vertically for 15 seconds and horizontally for 15 seconds (at 500 rpm). The plate is placed in the Cytomat 2C15 incubator. The reactions are stopped with three volumes of ice-cold stop solution*, using non-contact automated dispensing (Multidropcombi). The plates are centrifuged at 4oC and 3220 g for 20 min. The supernatant is diluted 1:1 with water (Biomek FXP robot, 80 µl supernatant + 80 µl water). From the start of pre-incubation of the cells and compounds to the end of the stop solution dispensing, all plate movements are carried out by the ORCA robot arm.

*Stop solution = Acetonitrile containing 0.8 % formic acid and 1 µM ofa volume marker (1µM 5, 5–diethyl–1, 3–diphenyl–2–iminobarbituric acid = no.39).

The test compounds are incubated with liver microsomes and NADPH in a 96-well plate at 37° C. An aliquot of the incubation mixture is taken after 0, 5, 15 and 45 min and the reaction is stopped with cold acetonitrile containing volume marker. The samples are centrifuge at 4000 rpm and supernatant is transferred and diluted with water in 96 well plates.

LC-MS/MS technique is used to determine substrate disappearance.

Clint human liver microsomes

Liver Microsomes: 1.0 mL of liver microsomes are thawed on ice at room temperature and diluted in 0.1M phosphate buffer pH=7.4 to concentration of 10 mg/mL.

NADPH 20 mM solution: 80 mg of NADPH is weighted and dissolved in 4.8 mL water to get 20 mM solution.

Test compounds stock solution: 1 mM solution of test and control compounds in DMSO is diluted with 50% acetonitrile to a concentration of 50 µM.

1 mMvolume marker stock solution MW=335 g/mol: 3.35 mg of no. 39 (5, 5-Diethyl-1, 3-diphenyl-2-iminobarbituric acid) is weighted and dissolved in 10 mLacetonitrile to give 1 mM solution. This solution is stored at 4 °C.

Stop solution: 0.5 mLof 1mM no.39 stock solution and 8 ml of formic acid is added to 1000 mL of acetonitrile.This solution is stored at 4 °C.

0.5 M K2HPO4*3 H2O MW=228.23 g/mol: 114 g of K2HPO4 *3 H2O is dissolved and diluted to 1000 mL with water. This solution is stored at 4 °C

0.5 M KH2PO4 MW=136 g/mol:68 gof KH2PO4 is dissolved and diluted to 1000 mL with water. This solution is stored at 4°C.

0.1 M Phosphate Buffer pH=7.4: 155 mL of 0.5 M K2HPO4 and 45 ml of 0.5 M KH2PO4 are diluted with water to 1000 mL. This solution is stored at 4°C.

Chromatographic system

A suitable chromatographic system is exemplified below.

Analytical column:Atlantis T3 C18, 3m, 2.1 x 30 mm

Gradient:4-95% acetonitrile in water (0.2% formic acid) in 1.6 minutes

LogD assay

A: 10 mM ammonium acetate in CH3CN/H2O 95/5

B: 10 mM ammonium acetate in CH3CN/H2O 5/95

(Mobile phase B is adjusted to pH 7.4 with ammonia and an equal amount ammonia is added to mobile phase A.)

Gradient: 99,9 % B for 0,17 min., linear gradient from 99,9 % B to 99,9 % A between 0,17 and 1.5 min., 99,9 % A for 0.26 min. Return to initial conditions and make sure the column is equilibrated before next injection. Flow rate: 1.0 ml/min for a 2.1 mm id column. For other dimensions, scale the flow rate appropriately.

Column: Waters ACQUITY UPLC® BEH C18 1.7 μm, 50 x 2.1 mm or column with similar selectivity.

Sample preparation: 100 μL 0.5mM in DMSO

Solubility experiments

From a mother plate of 8x12 wells, each well containing a minimum of 100 µl (5 or 10 mM) of a compound dissolved in DMSO, 10 µl is transferred to a precipitation plate and diluted with 3x330 µl buffer. Thus, the final samples contain 1% (v/v) DMSO.

The precipitation plate is shaken on a flat bed shaker for 16 hours, after which (400µl) of each solution is transferred to an 8x12 Whatman GF/B well filter placed on top of a vacuum manifold. Immediately after the filter is filled, vacuum is applied and the samples are filtered unto the filtration plate.

From the mother plate, compound dissolved in DMSO is also transferred to a standard plate, where the sample is diluted using an organic solvent suitable for the compound (ethanol, acetonitrile, etc.). One, two or three standards might be used, the concentration of the first standard is always set as a hundred-fold dilution from the mother plate concentration. Single samples are used for standards.

The samples are analysed with LC-MS.

MC1 receptor binding assay

Membrane preps are diluted to desired concentration in Assay Buffer. For over-expressing cell lines use 2µg protein per well (27 ug protein per ml, respectively).
74 µl of diluted membrane prep is added into the wells of the round bottom polypropylene plate.
1 µl of the compound/reference/total binding solutions is added from the compound dilution plate to the membrane containing plate in triplicate.
25 µl of tracer 125I-NDP-MSH is added to each well. Tracer is diluted in assay buffer to ~40,000CPM (final conc. ~70pM).
Plate is incubated in the Cytomat at 37°C for 1.5 hours.
Harvest filter plates are blocked with 1% PEI before reaction mix is added.
Reaction is filtered on FX vacuum onto the PEI soaked plates.
Plates are washed with ~1mL of cold 1X PBS wash buffer per well 4 times.
The plate is then dried at 50oC for 1 hr or at room temperature for 8h.
35 µl of Scintillation Cocktail is added to each well and the plate is incubated at room temperature for 1h.
The plate is sealed and measured by Microbeta counter for 125 I activity.

MC4 receptor binding assay

Membrane preps are diluted to desired concentration in Assay Buffer. For over-expressing cell lines use 0.75 µg protein per well (10 ug protein per ml, respectively).
74 µl of diluted membrane prep is added into the wells of the round bottom polypropylene plate.
1 µl of the compound/reference/total binding solutions is added from the compound dilution plate to the membrane containing plate in triplicate.
25 µl of tracer 125I-NDP-MSH is added to each well. Tracer is diluted in assay buffer to ~40,000CPM (final conc. ~70pM).
Plate is incubated in the Cytomat at 37°C for 1.5 hours.
Harvest filter plates are blocked with 1% PEI before reaction mix is added.
Reaction is filtered on FX vacuum onto the PEI soaked plates.
Plates are washed with ~1mL of cold 1X PBS wash buffer per well 4 times.
The plate is then dried at 50oC for 1 hr or at room temperature for 8h.
35 µl of Scintillation Cocktail is added to each well and the plate is incubated at room temperature for 1h.
The plate is sealed and measured by Microbeta counter for 125 I activity.

MC4 potency assay

HEK293TRexMC4 cells at 75% confluence are incubated with doxycycline (0.1ng/ml) at 37C/5%CO2 for 16-18 hrs before the start of the assay.

On the day of the assay cells are washed with PBS and harvested using Cell Dissociation Buffer, then centrifuged and re-suspended in the Assay Buffer.Cells are then counted and volume is adjusted to 5x104 cells per 200ul in Assay Buffer.

Cells are dispensed into cell plates byMultidrop at 198ul (5x104) cells/well and incubated 10 min in 370C

2uL of control, ref or compounds is transfered to the 96 wells cell plate by Beckman FX robotic system. Plates are gently shaken and incubated for 15 min in 37oC.

Reaction is stopped by adding 15ul of lysis buffer per well and incubate for 30 min at room temperature.

25ul of cells lysate from the assay plate is transferred into white half area plate (COSTAR 3693)

At the same time, 25ul of cAMP standard curve dilution is pipetted in triplicate into white half area plate and assayed with the stimulated cell samples

10µL of cAMP-d2 in Conjugate and Lysis buffer is added using MultidropCombi (The negative/cell negative controls gets 10µL Lysis buffer – No d2!)

10µL of anti cAMPcryptate in Conjugate and Lysis buffer is added using MultidropCombi to all assay wells

Incubation at RT for 1 hour.

Detection using Victor at 615 and 665nm.

Assay for inhibition of CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4

Stock solutions:

rCYP / NADPH (mM) / KPO4 pH 7.4 (M) / Substrate (µM) / Positive control (top conc, mM) / 100% inhibition conc (mM)
1A2 / 5 / 0.4 / CEC
400 / -naphtoflavone
0.25 / -naphtoflavone
0.25
2C8 / 5 / 0.4 / DBF
80 / Montelukast
10 / Montelukast
15
2C9 / 5 / 0.4 / MFC
1600 / Sulfaphenazole
22.5 / Sulfaphenazole
22.5
2C19 / 5 / 0.4 / MFC
1600 / Ticlopedine
15 / Ticlopedine
30
2D6 / 2 / 0.4 / MAMC
600 / Quinidine
0.25 / Quinidine
0.25
3A4 / 5 / 0.4 / BFC
1600 / Ketoconazole
1.5 / Ketoconazole
1.5

Substrates:

The substrates used in the assays were 3-Cyano-7-ethoxy-coumarin [CEC],Dibenzylfluorescein [DBF], 7-methoxy-4-trifluoromethylcoumarin [MFC], 7-methoxy-4- (aminomethyl)-coumarine [MAMC], and 7bensyloxy-4-trifluoromethyl-coumarin [BFC].

Chemicals / Company / Cat no / Amount / MW / Storage
CEC / SIGMA / C-2612 / 25 mg / 215.2 / RT
DBF / BD Gentest / 451755 / 25mg / 512.55 / -20C
MFC / SIGMA / T-3165 / 100 mg / 244.2 / RT
MAMC / BD Gentest / 451765 / 25 mg / 205 / -20C
BFC / BD Gentest / 451735 / 25 mg / 320 / -20C

Control inhibitors:

The reference substances with known inhibitory effect used as positive controls in the assays were -naphthoflavone, montelukast sodium, sulfaphenazole, ticlopedine, quinidine and ketoconazole.

The appropriate control inhibitor for each enzyme was delivered in serial dilutions in the same assay ready plates (black Greiner 384 well) from CM (100nL/well) as the test compounds. The dilutions were performed in DMSO giving 5 different concentrations. In addition, wells with high concentration of the control inhibitor and DMSO were also added to the plate for the measurement of 100% and 0% inhibition, respectively.

Chemicals / Company / Cat no / Amount / MW / Storage
-naphtoflavone / SIGMA / N-5757 / 1 g / 272.3 / 8C
MontelukastSodium / KB CHEMTRONICA / 151767-02-1 / 100mg / 608.18 / 8C
Sulfaphenazole / SIGMA / S-0758 / 25 g / 314.4 / RT
Ticlopidine / ICN Biomedicals Inc / 156522 / 300.2 / RT
Quinidine / SIGMA / Q-0750 / 5 g / 378.9 / RT
Ketoconazole / SIGMA / K-1003 / 100 mg / 531.4 / 8C

Master mix preparation for CYP 1A2:

CYP 1A2 / Enzyme / CEC pre-dilution / KPO4 (pH: 7.4) / dH2O
Stock conc. (µM) / 11.3 / 400 / 400000
Conc. in master mix (µM) / 0.002 / 12 / 124938
Final conc. (µM) / 0.002 / 10 / 100000
Volume (µl) / 3.87 / 547 / 5466 / 11484
Total volume (µl) / 17500 / Number of plates / 1

Master mix preparation for CYP 2C9:

CYP 2C9 / Enzyme / MFC pre-dilution / KPO4 (pH: 7.4) / dH2O
Stock conc. (µM) / 3 / 1600 / 400000
Conc. in master mix (µM) / 0.031 / 50 / 124938
Final conc. (µM) / 0.025 / 40 / 100000
Volume (µl) / 182 / 547 / 5466 / 11305
Total volume (µl) / 17500 / Number of plates / 1

Master mix preparation for CYP 2C19:

CYP 2C19 / Enzyme / MFC pre-dilution / KPO4 (pH: 7.4) / dH2O
Stock conc. (µM) / 4.9 / 1600 / 400000
Conc. in master mix (µM) / 0.050 / 50 / 124938
Final conc. (µM) / 0.040 / 40 / 100000
Volume (µl) / 178 / 547 / 5466 / 11309
Total volume (µl) / 17500 / Number of plates / 1

Master mix preparation for CYP 2D6:

CYP 2D6 / Enzyme / MAMC / KPO4 (pH: 7.4) / dH2O
Stock conc. (µM) / 3.7 / 600 / 400000
Conc. in master mix (µM) / 0.025 / 31 / 124938
Final conc. (µM) / 0.02 / 25 / 100000
Volume (µl) / 118 / 911 / 5466 / 11005
Total volume (µl) / 17500 / Number of plates / 1

Master mix preparation for CYP 3A4:

CYP 3A4 / Enzyme / BFC pre-dilution / KPO4 (pH: 7.4) / dH2O
Stock conc. (µM) / 7.3 / 1600 / 400000
Conc. in master mix (µM) / 0.019 / 25 / 124938
Final conc. (µM) / 0.015 / 20 / 100000
Volume (µl) / 44.9 / 273 / 5466 / 11716
Total volume (µl): / 17500 / Number of plates: / 1

Stop solution:

80 % acetonitrile, 20 % Tris Base 0.5 M
2M NaOH (for CYP2C8)

Method:

1Prepare substrate solutions, master mix and NADPH solutions.

2Add 40 µl master mix/well to assay ready compound plate containing test compounds and -naphtoflavone and shake plate (Multidropcombi).

3Read the background fluorescence in Tecan Safire 2 plate reader for identification of fluorescent test compounds on a separate plate.

4Pre-incubate the assay plate at 37°C for 10 minutes in the Thermo incubator.

5Add 10 µl NADPH (5mM) and shake plate (Multidropcombi).

6Incubate with shaking at 37°C 20 minutes (Thermo incubator).

7Add 25 µl stop solution (80 % ACN/0.1 M Tris Base) (Multidropcombi)

8Centrifuge the plate for ~1 minute (Eppendorf plate centrifuge)

9Read the fluorescence signal (Tecan Safire 2)