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By: Eric Chan & Jamie Yu March 13, 2014

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

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By: Eric Chan & Jamie Yu March 13, 2014

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  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

  6. 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

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

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

  9. 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)

  10. PXR CAR PXR CAR CAR PXR PXR CAR PXR

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

  12. PXR CAR PXR CAR CAR PXR PXR CAR PXR PXR

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

  14. PXR CAR PXR CAR CAR PXR PXR CAR PXR PXR CAR

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

  16. PXR CAR PXR CAR CAR PXR PXR CAR PXR PXR CAR CAR

  17. 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.

  18. 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

  19. PXR CAR PXR CAR PXR CAR CAR PXR

  20. PXR CAR PXR CAR PXR CAR CAR PXR PXR PXR PXR PXR CAR CAR CAR CAR

  21. 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

  22. 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

  23. PXR CAR PXR CAR PXR CAR CAR PXR PXR PXR PXR PXR CAR CAR CAR CAR

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

  25. PXR CAR PXR CAR PXR CAR CAR PXR PXR PXR PXR PXR CAR CAR CAR CAR

  26. 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

  27. 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

  28. 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.

  29. 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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