By: Eric Chan & Jamie Yu March 13, 2014. Introduction Nuclear Hormone Receptors Role as xenobiotic...
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Transcript of By: Eric Chan & Jamie Yu March 13, 2014. Introduction Nuclear Hormone Receptors Role as xenobiotic...
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.