1. By: Eric Chan & Jamie Yu March 13, 2014

2. Introduction Nuclear Hormone Receptors Role as xenobiotic sensors:
Pregname X Receptor (PXR)
Constitutive Androstane Receptor (CAR)
Well-conserved structures
Ligand binding  receptors translocate from the cytoplasm to the nucleus  regulate gene expression
Which are involved in the biotransformation of chemicals in a ligand-dependent manner

3. Goal To create: Strategy: Novel PXR and CAR-humanized mice
PXR- and CAR-KO mice
Panel of mice including all possible combinations of these genetic variants
Strategy:
Knockin
Knockout (KO)

4. Overview
Crosstalk
Def. One or more components of one signal transduction pathway affecting another
Ultimately, allowing the dissection of crosstalk between PXR and CAR in the response to drugs

5. Crosstalk Xenobiotics interact with PXR or CAR or both
Ligand binding leads to translocation
Retinoic X receptor (RXR) with PXR/CAR for heterodimerization
Bind to corresponding elements of target genes
Regulation of gene expression through PXR responsive element (PXRRE) and PB responsive element module (PBREM)
Gene regulation of drug-metabolizing enzymes or transporters
Between PXR and CAR
Xenobiotics interactions
Xenobiotics = foreign chemical substance found within an organism that is not normally naturally produced by or expected to be present within that organism
Ligand binding leads to translocation from cytoplasm into nucleus
Retinoic X Receptor (RXR) is used for heterodimerization
PXR and CAR mediate gene regulation through PXRRE
PXR responsive element

6. huPXR and huCAR mice generated by a knockin strategy
-expression of human receptors is controlled by the corresponding mouse promoters
-this strategy simultaneously deletes the endogenous gene function
Knockouts
All exons coding for functional domains of CAR were deleted in CAR KO and expression of PXR was prevented in PXR KO, both receptors represents complete KO
Kos for both receptors (PXR KO and CAR KO) were generated by crossing humanized mice to a ΦC31-deleter strain
ΦC31 recognition sites attB53 and attP50 in the targeting vectors

7. PXR-targeted mice
hPXR gene knocked in onto mPxr WT to generate PXR-targeted mice
Mouse exons: black and lowercase letters
Human exons: white with uppercase letters

8. CAR-targeted mice
mCar WT gene was replaced with the genomic coding region of hCAR to generate CAR-targeted mice

9. Evaluating mouse models
How did they evaluate mouse models?
Wild Type (mPXR/mCAR)
PXR
CAR
huCAR
Inducer (Drug)
CAR
huCAR KO (mPXR)
PXR
Western Blog Analysis for Phase 1 enzymes
(Cyp3a11 & Cyp2b10)
huPXR KO (mCAR)
CAR
huPXR
PXR

10. Between PXR and CAR
Xenobiotics interactions
Xenobiotics = foreign chemical substance found within an organism that is not normally naturally produced by or expected to be present within that organism
Ligand binding leads to translocation from cytoplasm into nucleus
Retinoic X Receptor (RXR) is used for heterodimerization
PXR and CAR mediate gene regulation through PXRRE
PXR responsive element

11. Inducer (Drug): Rifampicin (RIF)
PXR
Inducer’s target: Human PXR (huPXR)
Induction in huPXR mice (from low dosage)
Also found that with high dosage(60mg/kg), Induction in Wild Type Mouse (mPXR/mCAR)

12. PXR
CAR
PXR
CAR
CAR
PXR
CAR
PXR
Drug
Target
Wild Type (mPXR/mCAR)
huCAR
CAR KO (mPXR)
PXR KO (mCAR)
huPXR
RIF
Human PXR (huPXR)
Ø until high dosage Ø
Induction
PXR

13. Inducer: Dexamethasone (DEX)
PXR
Inducer’s target: Murine PXR (mPXR)
Induction in Wild Type (mPXR/mCAR)
Minimal induction for PXR KO and huPXR

14. PXR
CAR
PXR
CAR
CAR
PXR
CAR
PXR
Drug
Target
Wild Type (mPXR/mCAR)
huCAR
CAR KO (mPXR)
PXR KO (mCAR)
huPXR
RIF
Human PXR (huPXR)
Ø until high dosage Ø
Induction
DEX
Murine PXR (mPXR)
Ø (Slightly on high dose)
PXR
PXR

15. Inducer: CITCO CAR Inducer’s target: Human CAR (huCAR)
Induction for huCAR
No significant induction for Wild Type, CAR KO, PCR KO or huPXR

16. Ø (Slightly on high dose)
PXR
CAR
PXR
CAR
CAR
PXR
CAR
PXR
Drug
Target
Wild Type (mPXR/mCAR)
huCAR
CAR KO (mPXR)
PXR KO (mCAR)
huPXR
RIF
Human PXR (huPXR)
Ø until high dosage Ø
Induction
DEX
Murine PXR (mPXR)
Ø (Slightly on high dose)
CITCO
Human CAR (huCAR)
Ø until high dose
PXR
PXR
CAR

17. Inducer: TCPOBOP CAR Inducer’s target: Murine CAR (mCAR)
Induction in Wild Type, PXR KO
No significant induction in huCAR, CAR KO

18. Ø (Slightly on high dose)
PXR
CAR
PXR
CAR
CAR
PXR
CAR
PXR
Drug
Target
Wild Type (mPXR/mCAR)
huCAR
CAR KO (mPXR)
PXR KO (mCAR)
huPXR
RIF
Human PXR (huPXR)
Ø until high dosage Ø
Induction
DEX
Murine PXR (mPXR)
Ø (Slightly on high dose)
CITCO
Human CAR (huCAR)
Ø until high dose
TCPOBOP
Murine CAR (mCAR)
PXR
PXR
CAR
CAR

19. What is the point of that?
Summary:
Confirmed that huPXR & huCAR human receptors in mice are functional
Both huPXR and huCAR show the expected humanized profile (according to established papers) of interaction with different inducers
In short, mice models were good.

20. Now what?
They created a panel of mouse lines of all the possibilities …
So what are the possibilities?
Why?
in order to determine whether another drug (Phenobarbital) targets:
PXR
CAR
Both PXR and CAR

21. PXR
CAR
PXR
CAR
CAR
CAR
PXR
PXR
Drug
Which P450?
Wild Type (mPXR/mCAR)
huCAR
PXR KO (mCAR)
CAR KO (mPXR)
Phenobarbital
Cyp3a11
(PB)
Cyp2b10

22. PXR
CAR
PXR
CAR
CAR
CAR
PXR
PXR
Drug
Which P450?
Wild Type (mPXR/mCAR)
huCAR
PXR KO (mCAR)
CAR KO (mPXR)
Phenobarbital
Cyp3a11
(PB)
Cyp2b10
PXR
PXR
PXR
PXR
CAR
CAR
CAR
CAR
Drug
Which P450?
huPXR/huCAR
PXR KO/
CAR KO
huPXR/
PXR KO/ huCAR
Phenobarbital
Cyp3a11
(PB)
Cyp2b10

23. Now what?
They created a panel of mouse lines of all the possibilities …
So what are the possibilities?
Why?
in order to determine whether another drug (Phenobarbital) targets:
PXR
CAR
Both PXR and CAR

24. Inducer: Phenobarbital (PB)
Before we had excluded the cytochrome type for simplicity’s sake. But now:
For Cyp3a11:
Slight induction in all except for CAR KO
For Cyp2b10:
Induction in all except for CAR KO
Now both, because for more accuracy.

25. PXR
CAR
PXR
CAR
CAR
CAR
PXR
PXR
Drug
Which P450?
Wild Type (mPXR/mCAR)
huCAR
PXR KO (mCAR)
CAR KO (mPXR)
Phenobarbital
Cyp3a11
Slight Indu. Ø
(PB)
Cyp2b10
Induction
PXR
PXR
PXR
PXR
CAR
CAR
CAR
CAR
Drug
Which P450?
huPXR/huCAR
PXR KO/
CAR KO
huPXR/
PXR KO/ huCAR
Phenobarbital
Cyp3a11
(PB)
Cyp2b10

26. Inducer: Phenobarbital (PB)
For Cyp3a11:
Slight induction in HuPXR/HuCAR & PXR KO/huCAR
For Cyp2b10:
Induction in in HuPXR/HuCAR & PXR KO/huCAR

27. PXR
CAR
PXR
CAR
CAR
CAR
PXR
PXR
Drug
Which P450?
Wild Type (mPXR/mCAR)
huCAR
PXR KO (mCAR)
CAR KO (mPXR)
Phenobarbital
Cyp3a11
Slight Indu. Ø
(PB)
Cyp2b10
Induction
PXR
PXR
PXR
PXR
CAR
CAR
CAR
CAR
Drug
Which P450?
huPXR/huCAR
PXR KO/
CAR KO
huPXR/
PXR KO/ huCAR
Phenobarbital
Cyp3a11
Slight Indu. Ø
(PB)
Cyp2b10
Induction

28. What does that mean?
huPXR is not able to compensate for the lost of CAR activity
Phenobarbital (PB) is described as a PXR and CAR activator under vitro analysis
But our in vivo studies suggests that Phenobarbital (PB) was only mediated by CAR.
PXR
CAR

29. Importance Therefore, able to use these findings clinically
Drug metabolizing enzymes and process
Limitations on previous methods
Depending on the cell line or primary culture system used
Need for promoter/enhancer sequence of the reporter construct
Complex interactions of an in vivo system cannot be predicted in the absence of cell lines with significant hepatic functions

30. What’s next?
They want to create mouse panels that includes human phase 1, phase 2, and phase 3 genes onto the hCAR/hPXR background.

31. Take Home Message
Created a panel to reflect more accurately how drugs might react in humans.
This was the first humanize mouse models using the knockin and knockout genes to study the pharmacokinetics of drugs.