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By: Eric Chan & Jamie Yu March 13, 2014. Introduction Nuclear Hormone Receptors Role as xenobiotic sensors: Pregname X Receptor (PXR) Constitutive Androstane.

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Presentation on theme: "By: Eric Chan & Jamie Yu March 13, 2014. Introduction Nuclear Hormone Receptors Role as xenobiotic sensors: Pregname X Receptor (PXR) Constitutive Androstane."— Presentation transcript:

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

3 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)

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

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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) Inducer (Drug) huCAR huCAR KO (mPXR) huPXR KO (mCAR) huPXR PXR CAR PXR CAR PXR Western Blog Analysis for Phase 1 enzymes (Cyp3a11 & Cyp2b10)

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11 Inducer (Drug): Rifampicin (RIF) Inducers 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

12 DrugTargetWild Type (mPXR/mCAR) huCARCAR KO (mPXR) PXR KO (mCAR) huPXR RIFHuman PXR (huPXR) Ø until high dosage ØInduction PXR CAR PXR CARPXR CAR PXR

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

14 DrugTargetWild Type (mPXR/mCAR) huCARCAR KO (mPXR) PXR KO (mCAR) huPXR RIFHuman PXR (huPXR) Ø until high dosage ØInduction DEX Murine PXR (mPXR) InductionØØ (Slightly on high dose) PXR CAR PXR CARPXR CAR PXR

15 Inducer: CITCO Inducers target: Human CAR (huCAR) – Induction for huCAR – No significant induction for Wild Type, CAR KO, PCR KO or huPXR CA R

16 DrugTargetWild Type (mPXR/mCAR) huCARCAR KO (mPXR) PXR KO (mCAR) huPXR RIFHuman PXR (huPXR) Ø until high dosage ØInduction DEX Murine PXR (mPXR) InductionØØ (Slightly on high dose) CITCOHuman CAR (huCAR) Ø until high dose InductionØØØ PXR CAR PXR CARPXR CAR PXR

17 Inducer: TCPOBOP Inducers target: Murine CAR (mCAR) – Induction in Wild Type, PXR KO – No significant induction in huCAR, CAR KO CA R

18 DrugTargetWild Type (mPXR/mCAR) huCARCAR KO (mPXR) PXR KO (mCAR) huPXR RIFHuman PXR (huPXR) Ø until high dosage ØInduction DEX Murine PXR (mPXR) InductionØØ (Slightly on high dose) CITCOHuman CAR (huCAR) Ø until high dose InductionØØØ TCPOBOPMurine CAR (mCAR) InductionØØ PXR CAR PXR CARPXR CAR PXR 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 DrugWhich P450? Wild Type (mPXR/mCA R) huCARPXR KO (mCAR) huCARCAR KO (mPXR) PhenobarbitalCyp3a11 (PB)Cyp2b10 PXR CAR PXR CAR PXR CAR PXR

22 DrugWhich P450? huPXR/huCARPXR KO/ CAR KO huPXR/ CAR KO PXR KO/ huCAR PhenobarbitalCyp3a11 (PB)Cyp2b1 0 DrugWhich P450? Wild Type (mPXR/mCA R) huCARPXR KO (mCAR) huCARCAR KO (mPXR) PhenobarbitalCyp3a11 (PB)Cyp2b10 PXR CAR PXR CAR PXR CAR PXR CAR PXR CAR PXR CAR PXR CAR PXR

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 simplicitys sake. But now: For Cyp3a11: – Slight induction in all except for CAR KO For Cyp2b10: – Induction in all except for CAR KO

25 DrugWhich P450? huPXR/huCARPXR KO/ CAR KO huPXR/ CAR KO PXR KO/ huCAR PhenobarbitalCyp3a11 (PB)Cyp2b1 0 DrugWhich P450? Wild Type (mPXR/mCA R) huCARPXR KO (mCAR) huCARCAR KO (mPXR) PhenobarbitalCyp3a11 Slight Indu. Ø (PB)Cyp2b10Induction Ø PXR CAR PXR CAR PXR CAR PXR CAR PXR CAR PXR CAR PXR CAR PXR

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 DrugWhich P450? huPXR/huCARPXR KO/ CAR KO huPXR/ CAR KO PXR KO/ huCAR PhenobarbitalCyp3a11Slight Indu.ØØ (PB)Cyp2b1 0 InductionØØ DrugWhich P450? Wild Type (mPXR/mCA R) huCARPXR KO (mCAR) huCARCAR KO (mPXR) PhenobarbitalCyp3a11 Slight Indu. Ø (PB)Cyp2b10Induction Ø PXR CAR PXR CAR PXR CAR PXR CAR PXR CAR PXR CAR PXR CAR PXR

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. CAR PXR

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 Whats 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.


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