By: Eric Chan & Jamie Yu March 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 Which are involved in the biotransformation of chemicals in a ligand-dependent manner

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)

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

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

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

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

CAR-targeted mice mCar 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) 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

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

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)

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

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

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

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

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

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

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

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

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 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

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 Now both, because for more accuracy.

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

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

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

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

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.