Hepatocyte Protocols

1. Fresh/Inducible Cryopreserved Human Hepatocyte Induction Assays
   
2. Cryopreserved Hepatocyte Metabolic Stability Assay
   
   

The cellular content of cytochromes P450 can be induced several fold by pre-treatment with drugs and other xenobiotics. Drug mediated induction of P450 can result in increased metabolism of other drugs or itself, leading to potentially harmful drug interactions and/or drug tolerance. Freshly isolated human hepatocytes provide an excellent in vitro means for predicting P450 induction potential by drug candidates.

The following protocol describes a method for treating plated human hepatocytes with "classical" P450 inducers (i.e. Rifampicin-CYP3A4, 2C9/19; Phenobarbital-CYP2B6, 3A4; and ß-Naphthoflavone-CYP1A2), and analysis of results. The same general protocol can be followed when testing the induction potential of "new chemical entities" (NCE).

Table 1: Chemicals and Suppliers

Table 2: P450 Inducers and Substrates/Solvents and Concentrations:

* Specific Activity of 10 mM stock: 5 to 7 mCi/mmole

Other Material and Equipment

A. Bio-safety hood
B. Incubator with 37°C and 95% air/ 5% C02 at atmospheric level capacity
C. Basic cell culture equipment

*Hepato-STIM, BD-Biosciences and Gentest are trademarks of Becton-Dickinson and Co.

Hepatocytes:
Plated Human Hepatocytes/BD-Biocoat Collagen I Microplate

a). Cells in Multi-well plate format
6-well BD-Biocoat plates (Cat. No. 454406)
12-well BD-Biocoat plates (Cat. No. 454412)
24-well BD-Biocoat plates (Cat. No. 454424)
96-well BD-Biocoat plates (Cat. No. 454496)

Procedure:
1. Unpack the plated hepatocytes. Follow enclosed instructions for handling requirements and maintenance until ready for further testing.

2. Incubate the plated hepatocytes for 2 days in a humidified 37°C incubator with 95%air/ 5% C02 atmosphere to allow for recovery from shipping.

3. Prepare the chemical inducers (RIF, ß-NF, and PB), solvent vehicle controls (DMSO for RIF and ß-NF, and H2O for PB) and any test samples in Hepato-STIM media (Table 3). The Hepato-STIM/inducer preparation should be prepared fresh on the day of use.

Note: Whenever DMSO is used to dissolve test compound the final DMSO concentration should not exceed 0.1%.

Table 3: Treatment Schedule and Inducer Concentrations

4. Aspirate the media from the well and replace the media containing the desired treatment condition. Use the volumes shown below (Table 4).

Table 4: Media Volumes and Cell Number/Well

5. Return the cells to the incubator for 24 hours.

6. Repeat steps 4 and 5 twice so that the hepatocytes have a total of three exposures to the treatment conditions over a 72 hours time period (Table 4).

Sample Analysis for Enzyme Induction

There are currently a number of methods for analyzing samples for P450 induction, e.g. protein levels by immuno blots, mRNA levels by Northern blots or RT-PCR, and P450 specific probe substrate activity.
The following are protocols for measuring specific P450 activities using probe substrates. Quantification of the P450-dependent metabolites relies on HPLC analysis.

1. Dissolve the P450 substrate in the appropriate volume of KHB buffer to give a final concentration as shown below (Table 5).

Table 5: P450 Substrates, Activities, and Working Stock Concentrations

2. Remove the cell culture media and replace it with the same volume of substrate/KHB buffer mix.

3. The cells should be incubated in a 37°C incubator (with CO2) for approximately 30 minutes to 1 hour. Additional time points may be included to establish linear conditions. If longer time points are used the substrates should be dissolved in Hepato-STIM media to insure cell survival.

4. At the end of the incubation period the assay is stopped by adding an appropriate volume of stop solution to the well. Table 6 shows suitable stop solutions and volumes that are compatible with HPLC analysis.

5. Analyze samples by HPLC. HPLC methods are available on the Gentest web site or in the Gentest catalog. Activity values can be reported as pmol product/min/mg of cell lysate or pmol product/min/E6 cells. Induction potential of the test compound is determined by dividing the activity of the treated group vs. the vehicle control group.

Table 6: Reaction-Stop Solutions

CRYOPRESERVED HEPATOCYTE METABOLIC STABILITY ASSAY <top>

Isolated hepatocytes contain all the enzymes and co-factors needed for phase I and phase II drug metabolism, making them an excellent in vitro model for assessment of drug metabolic stability and metabolite profiling. The following protocol describes a method for evaluating the metabolism of a test compound using cryopreserved human hepatocytes. A positive control assay is included in the protocol for the Phase I (P450) and Phase II (uridine glucuronosyl transferase-UGT and sulfer transferase-ST) metabolism of 7-Ethoxycoumarin. 7-Ethoxycoumarin is O-deethylated by P450, and conjugated by UGTs, and, to a lesser extent ST, (about 15:1 glucuronidation vs. sulfation) in human hepatocytes. The reverse is true for rat hepatocytes where sulfation occurs to a much greater extent than glucuronidation.

Chemicals and Suppliers

Materials:

A. Cryopreserved Human or Rat Hepatocytes (*BD-Gentest)
B. Hepatocyte Purification Kit (*BD-Gentest, Catolog No.454500)
C. 37ºC water bath
D. Low speed centrifuge
E. Biosafety hood
F. Vacuum pump
G. Incubator at 37°C and atmospheric level capacity
H. Multi well plates (96, 48, 24, 12, or 6 well formats, *BD-Falcon)

Solutions:

A. Krebs-Henseleit Buffer (KHB) (Sigma Chem. Co., Catalog No. K-3753). Prepared according to instructions provided with KHB buffer.
B. 1M Fructose
C. 300mM Glycine
D. 0.1 M 7-EC (dissolved in DMSO)
E. 7-EC metabolite standards dissolved in H2O to a final concentration of 200 uM
F. Test Compound (dissolved in DMSO or appropriate solvent)

Procedure:

1. Prepare the test compound by dissolving directly into KHB buffer*, or by preparing a stock solution in an organic solvent and diluting into KHB buffer. If an organic solvent is used to dissolve the test compounds, than the stock concentration should be high enough such that the final organic solvent concentration in the incubation is at a minimum. For example, final methanol and acetonitrile concentrations should be below 1%, while final DMSO concentrations should be below 0.2%.

Note*: For optimal results with cryopreserved rat hepatocytes, prepare KHB buffer with 10mM fructose and 3mM glycine.

2. For test compound stocks dissolved in organic solvent, a convenient step is to further dilute the test compound in KHB buffer to a make a 2X concentrate (i.e. if the final assay concentration is to be 100 uM, than the 2X stock in KHB buffer would be 200 uM).

3. Prepare the positive control stock in DMSO as described above. The 7-EC/DMSO stock should be 0.1 M. The 2X concentrate of 7-EC in KHB buffer is 200 uM, making the final assay concentration 100 uM.

4. Follow the hepatocyte thaw procedure and percoll purification procedure enclosed with the BD Gentest™ Hepatocyte Purification Kit in order to successfully recover the hepatocytes.

5. Resuspend the cells in KHB buffer (or KHB with 10mM fructose, 3mM glycine) at a final concentration of about 0.5 x 106 cells per ml. A desirable final cell concentration for most applications is 0.25 x 106 cells/ml.

6. Mix equal volumes of test compound in KHB and resuspend hepatocytes. The final volume will depend on the incubation vessel. For example, use 125 ul final volume per well in a 96 well plate and 200 ul per well in a 24 well plate.

7. Incubate in a humidified, 37°C, atmospheric pressure incubator for the desired length of time (e.g. 60 min.). Multiple time points (10 min up to 90 min) are recommended for determining metabolic stability or metabolite profile of a test compound.

8. Quench the incubation with an equal volume of acetonitrile or other stop solutions that are compatible with the particular analytical method. The amount of stop solution added may also vary depending on the analytical method.

9. The positive control incubation should be quenched with ZnSO4 and BaOH. The volume of each quench solution added should equal to 25% of the assay volume (e.g. 50 ul of each per 200 ul incubation volume).
Note: The ZnSO4 solution should always be added prior to the BaOH solution.

10. Transfer to eppendorf tubes and centrifuge at 14,000 rpm for 3 min. to pellet debris.

11. Remove the supernatant for immediate analysis (e.g. LC/MS, HPLC) or store samples at -20°C.

12. Analytical Method for Positive Control Assay (HPLC Method).

a) A Water 2690 or equivalent HPLC instrument should be used for separation of metabolites of 7-EC.

b) The HPLC column should be a C-18, 4.6 x 250 mm (5µm). We recommend a Zorbax C-18 column (HP-Cat. No. 880975-902). The column temperature should be set to 45°C. The HPLC flow rate is 1 ml/min.

c) Metabolite peaks are measured by UV detection at 320 nm or fluorescent detection of 320/380, gain of 100.

d) A typical injection volume is 100 ul of assay supernatant.

e) The HPLC mobile phases consist of the following: Mobile Phase A: 0.1% Trifluoroacetic Acid in H2O, Mobile Phase B: 100% Methanol, Mobile Phase B: 0.1% Trifluoroacetic Acid in acetonitrile

f) Initial HPLC conditions are 100% Mobile Phase A. Upon sample injection, Mobile Phase C is increased to 10% over a 15 min period, followed by an increase to 60% over 10 min, and a final increase to 60% over a 10 min period to elute the 7-EC parent compound. The entire HPLC run time is 30 min.

g) Metabolites are quantitated by comparison to the peak areas of known amounts of authentic metabolite standards.