Purpose

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-Naphthoavone-

CYP1A2) and analysis of results. The same general protocol can be followed when testing the

induction potential of new chemical entities (NCEs).

Table 1: Chemicals and Suppliers

Chemical Reagent Supplier (Cat. No.)

Rifampicin (RIF) Sigma Chemical Co. (R-3501)

ß-naphthoavone (ß-NF) Sigma Chemical Co. (N-3633)

Phenobarbital (PB) Sigma Chemical Co. (P-5178)

Corning Hepatocyte Culture Media Kit Corning Life Sciences (355056)

Dimethyl Sulfoxide (DMSO) JT Baker (T15599)

KHB Buffer Sigma Chemical Co. (K-3753)

Phenacetin (CYP1A2 substrate) Sigma Chemical Co. (A-2375)

Testosterone (CYP3A4 substrate) Sigma Chemical Co. (T-1500)

Diclofenac (CYP2C9 substrate) Sigma Chemical Co. (D-6899)

[14C]S-Mephenytoin (CYP2C19/2B6 substrate) GE Life Sciences (CFA-763)

S-Mephenytoin (CYP2C19/2B6 substrate) Corning Life Sciences (451032)

Table 2: P450 Inducers and Substrates/Solvents and Concentrations

Chemical Stock Concentration Solvent Final Working Concentration

Rifampicin (RIF) 25 mM DMSO 20 µM

ß-Naphthoavone (ß-NF) 25 mM DMSO 20 µM

Phenobarbital (PB) 0.2 M PBS 2 mM

Testosterone 0.1 M DMSO 200 µM

Diclofenac 0.1 M DMSO 100 µM

Phenacetin 0.1 M DMSO 100 µM

*[14C]S-Mephenytoin 10 mM ACN 100 µM

* Specic Activity of 10 mM stock: 5 to 7 mCi/mmol.

Corning

Fresh Human Hepatocyte

Cytochrome P450 Induction Assays

Protocol - Hepatocyte Assay Methods

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.

Other Material and Equipment:

w Biosafety hood

w Incubator with 37°C and 95% air/5% CO

at atmospheric level capacity

w Basic cell culture equipment

Fresh Plated Hepatocytes:

Plated Human Hepatocytes/Corning® BioCoat™ Collagen I microplates

w 6 well Corning BioCoat Collagen I plates (Cat. No. 454406)

w 12 well Corning BioCoat Collagen I plates (Cat. No. 454412)

w 24 well Corning BioCoat Collagen I plates (Cat. No. 454424)

w 48 well Corning BioCoat Collagen I plates (Cat. No. 454425)

w 96 well Corning BioCoat Collagen I 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 1-2 days in a humidified 37°C incubator with

95% air/5% CO

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 PBS for PB) and any test compounds (Table 3). The media/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

Chemical Inducer Working Concentration P450 Total Treatment Time

Rifampicin 20 µM 3A4, 2C9, 2C19 72 hours

PB 2 mM 2B6 72 hours

ß-NF 20 µM 1A2 72 hours

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

Approximate Cell number/Well

Multiwell Plate Volume of Media (100% conuent)

6 well 2 mL 14.4 x 10

12 well 800 µL 6 x 10

24 well 400 µL 3 x 10

48 well 145 µL 1.1 x 10

96 well 50 µL 0.42 x 10

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 3 day exposures to the

treatment conditions over a 72 hour time period (Table 4).