In vitro test system to evaluate xenobiotics as immune-modulators of drug transport and metabolism in human hepatocytes

1. A in vitro method of evaluating the effect of a xenobiotic on drug metabolism in hepatocytes, wherein said xenobiotic is a small molecule drug, said method comprising:

providing a first culture of hepatocytes;

providing a portion of an in vitro xenobiotic-stimulated biological sample by:

providing a first culture of a biological sample;

exposing said first biological sample to said xenobiotic small molecule drug in vitro for a period of time to yield said in vitro xenobiotic-stimulated biological sample; and

separating said portion of said in vitro xenobiotic-stimulated biological sample;

transferring said portion of said in vitro xenobiotic-stimulated biological sample to said first culture of hepatocytes; and

detecting the activity, expression, or combination thereof of a biomarker in said hepatocytes to thereby evaluate the effect of said in vitro xenobiotic on said drug metabolism in said hepatocytes.

2. The method of claim 1 , wherein said exposing comprises incubating said first biological sample with said xenobiotic for at least about 12 hours prior to said separating.

3. The method of claim 1 , wherein said biological sample is selected from the group consisting of whole blood, bone marrow, and isolated peripheral blood mononuclear cells.

4. The method of claim 1 , wherein said biological sample is whole blood and wherein said portion of said in vitro xenobiotic-stimulated biological sample is xenobiotic-stimulated plasma.

5. The method of claim 1 , wherein said portion of said in vitro xenobiotic-stimulated biological sample comprises a plurality of cytokines.

6. The method of claim 1 , further comprising incubating said portion of said in vitro xenobiotic-stimulated biological sample and said first culture of hepatocytes for at least about 24 hours prior to said detecting.

7. The method of claim 1 , wherein said detecting comprises incubating said hepatocytes with an enzyme marker substrate and measuring levels of said substrate or metabolites of the same.

8. The method of claim 1 , wherein said detecting comprises isolating microsomes from said hepatocytes and measuring the activity of drug-metabolizing enzymes in said microsomes.

9. The method of claim 1 , wherein said detecting comprises isolating total RNA from said hepatocytes and measuring enzyme mRNA levels of said drug-metabolizing enzymes.

10. The method of claim 1 , wherein said culture of hepatocytes is a co-culture of hepatocytes and Kupffer cells.

11. The method of claim 1 , wherein said hepatocytes are pooled hepatocytes.

12. The method of claim 1 , further comprising:

providing a second culture of hepatocytes;

exposing said second culture of hepatocytes directly to said xenobiotic; and

detecting the activity, expression, or combination thereof of a biomarker in said second culture of hepatocytes.

13. The method of claim 12 , further comprising comparing said activity and/or expression of said biomarker in said second culture of hepatocytes to said activity and/or expression of said biomarker in said first culture of hepatocytes.

14. The method of claim 1 , wherein said xenobiotic is dispersed in a carrier prior to said exposing.

15. The method of claim 14 , further comprising:

providing a third culture of hepatocytes;

exposing said third culture of hepatocytes directly to said carrier; and

detecting the activity, expression, or combination thereof of a biomarker in said third culture of hepatocytes.

16. The method of claim 15 , further comprising comparing said activity and/or expression of said biomarker in said third culture of hepatocytes to said activity and/or expression of said biomarker in said first culture of hepatocytes.

17. The method of claim 14 , further comprising:

providing a second culture of a biological sample and a fourth culture of hepatocytes;

exposing said second biological sample to said carrier for a period of time to yield a carrier-stimulated biological sample;

transferring a portion of said carrier-stimulated biological sample to said fourth culture of hepatocytes; and

detecting the activity, expression, or combination thereof of a biomarker in said fourth culture of hepatocytes.

18. The method of claim 17 , further comprising comparing said activity and/or expression of said biomarker in said fourth culture of hepatocytes to said activity and/or expression of said biomarker in said first culture of hepatocytes.

19. The method of claim 1 , further comprising:

providing a third culture of a biological sample and a fifth culture of hepatocytes;

exposing said third biological sample to a positive control compound with known cytokine-stimulating effects for a period of time to yield a positive control-stimulated biological sample;

transferring a portion of said positive control-stimulated biological sample to said fifth culture of hepatocytes; and

detecting the activity, expression, or combination thereof of a biomarker in said fifth culture of hepatocytes.

20. The method of claim 19 , further comprising comparing said activity and/or expression of said biomarker in said fifth culture of hepatocytes to said activity and/or expression of said biomarker in said first culture of hepatocytes.