By: Eric Chan & Jamie YuMarch 13, 2014

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

Goal

• To create:– 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)

Overview

• CrosstalkDef. 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

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

PXR-targeted mice

• hPXR gene knocked in onto mPxr WT to generate PXR-targeted mice

Mouse exons: black and lowercase lettersHuman exons: white with uppercase letters

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

Evaluating mouse models

• How did they evaluate mouse models?

Wild Type (mPXR/mCAR)

Inducer (Drug) huCAR

huCAR KO (mPXR)

huPXR KO (mCAR)

huPXR

PXRCAR

CAR

PXR

CAR

PXR

Western Blog Analysis for Phase 1 enzymes(Cyp3a11 & Cyp2b10)

Inducer (Drug): Rifampicin (RIF)

• 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)

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

PXRCAR

CARPXR CAR PXR

PXR

CAR PXR

Inducer: Dexamethasone (DEX)

• Inducer’s target: Murine PXR (mPXR)– Induction in Wild Type (mPXR/mCAR)– Minimal induction for PXR KO and huPXR

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)

Induction Ø Ø (Slightly on high dose)

PXRCAR

CARPXR CAR PXR

PXR

PXR

CAR PXR

Inducer: CITCO

• Inducer’s target: Human CAR (huCAR)– Induction for huCAR– No significant induction for Wild Type, CAR KO, PCR

KO or huPXR

CAR

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)

Induction Ø Ø (Slightly on high dose)

CITCO Human CAR (huCAR)

Ø until high dose

Induction Ø Ø Ø

PXRCAR

CARPXR CAR PXR

PXR

PXR

CAR

CAR PXR

Inducer: TCPOBOP

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

CAR

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)

Induction Ø Ø (Slightly on high dose)

CITCO Human CAR (huCAR)

Ø until high dose

Induction Ø Ø Ø

TCPOBOP Murine CAR (mCAR)

Induction Ø Ø Induction

PXRCAR

CARPXR CAR PXR

PXR

PXR

CAR

CAR PXR

CAR

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.

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

Drug Which P450?

Wild Type (mPXR/mCAR)

huCAR PXR KO (mCAR)

huCAR CAR KO (mPXR)

Phenobarbital Cyp3a11

(PB) Cyp2b10

PXRCAR

CARPXRCAR PXR

CARPXR

Drug Which P450?

huPXR/huCAR PXR KO/CAR KO

huPXR/CAR KO

PXR KO/ huCAR

Phenobarbital Cyp3a11

(PB) Cyp2b10

Drug Which P450?

Wild Type (mPXR/mCAR)

huCAR PXR KO (mCAR)

huCAR CAR KO (mPXR)

Phenobarbital Cyp3a11

(PB) Cyp2b10

PXRCAR

CARPXRCAR PXR

CARPXR

CARPXR

CARPXR

CARPXR

CARPXR

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

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

Drug Which P450?

huPXR/huCAR PXR KO/CAR KO

huPXR/CAR KO

PXR KO/ huCAR

Phenobarbital Cyp3a11

(PB) Cyp2b10

Drug Which P450?

Wild Type (mPXR/mCAR)

huCAR PXR KO (mCAR)

huCAR CAR KO (mPXR)

Phenobarbital Cyp3a11 Slight Indu. Slight Indu. Slight Indu. Slight Indu.

Ø

(PB) Cyp2b10 Induction Induction Induction Induction Ø

PXRCAR

CARPXRCAR PXR

CARPXR

CARPXR

CARPXR

CARPXR

CARPXR

Inducer: Phenobarbital (PB)

• For Cyp3a11:– Slight induction in HuPXR/HuCAR & PXR KO/huCAR

• For Cyp2b10:– Induction in in HuPXR/HuCAR & PXR KO/huCAR

Drug Which P450?

huPXR/huCAR PXR KO/CAR KO

huPXR/CAR KO

PXR KO/ huCAR

Phenobarbital Cyp3a11 Slight Indu. Ø Ø Slight Indu.

(PB) Cyp2b10

Induction Ø Ø Induction

Drug Which P450?

Wild Type (mPXR/mCAR)

huCAR PXR KO (mCAR)

huCAR CAR KO (mPXR)

Phenobarbital Cyp3a11 Slight Indu. Slight Indu. Slight Indu. Slight Indu.

Ø

(PB) Cyp2b10 Induction Induction Induction Induction Ø

PXRCAR

CARPXRCAR PXR

CARPXR

CARPXR

CARPXR

CARPXR

CARPXR

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.

CARPXR

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

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.

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.