mechanisms of hepatic enzyme induction in humans and how to assess it in vitro edward l. lecluyse,...
TRANSCRIPT
Mechanisms of Hepatic Enzyme Induction in Humans and How to
Assess It In Vitro
Edward L. LeCluyse, Ph.D.Chief Scientific Officer, CellzDirect, Inc.
November 2004
Pertinent Questions
If a NME’s induction effect on CYP3A4 in vitro is NEGATIVE, then is it acceptable to NOT recommend any in vivo studies with substrates of CYP3A, CYP2C9, CYP2B6 and CYP2C19.
– YES or NO?
If the in vitro induction (increase in enzyme activity) is more than 40% of the positive control (e.g., rifampin), then there IS a need to recommend an in vivo induction study.
– YES or NO?
Induced PlasmaInduced PlasmaDrugDrug CYPsCYPs Conc (µm)Conc (µm) NRNR Drugs affected Drugs affected in vivoin vivo
CarbamazepineCarbamazepine 3A, 2B 20-40CAR Praziquantel, Itraconazole, Cyclosporin A
SJWSJW 3A, 2C 0.2 AhR, PXR Indinavir, Digoxin, Cyclosporin, Theophylline
PhenytoinPhenytoin 2C, 3A, 2B >10 CAR Warfarin, Praziquantel, Quinidine, Cyclosporin A
PhenobarbitalPhenobarbital 2C, 3A, 2B 40-130 CAR, PXR Warfarin, Cyclosporin A
RifampinRifampin 2C, 3A, 2B 14 PXR Tolbutamide, WarfarinCyclosporin, Oral contraceptives
TroglitazoneTroglitazone 3A, 2B 7 PXR Cyclosporin A, Terfenadine
AvasimibeAvasimibe 2C, 3A, 2B 1-6 PXR Midazolam, Warfarin, Digoxin
Enzyme Induction in Humans
Inducible P450 Enzymes in Human Liver
P450 Enzyme InducersCYP1A2 Aromatic hydrocarbons, cigarette smoke,
cruciferous vegetables, charbroiled meat
CYP2A6 Anticonvulsants, DEX
CYP2B6 phenytoin, PB, RIF
CYP2C8 Anticonvulsants, rifampin
CYP2C9 Rifampin, Anticonvulsants
CYP2C19 Rifampin, Anticonvulsants
CYP3A4 CLZ, Rifampin, Anticonvulsants
CYP2E1 Isoniazid, alcohol
CYP2D6 Non-inducibleCYP4A11 Non-inducible (?)
CYP2B, CYP3A, CYP1A, CYP2A, CYP2Cs, OATP2UGT1A1, MRP2, SULT1A1
CYP1A, UGT1A1, SULT1A1
CAR RXR
XREM / PBREM
AhR ARNT
XRE / DRE
PXR RXR
XREM / PXRE
CYP3A, CYP2B, CYP2Cs,CYP7A, MDR1, MRP2, OATP2, GSTA-2, UGT1A1,AldHs, Carboxyesterase 2, 3
Strong Mod Weak
CLZ CMZ DTBARIF SPZRITPHNPB
Strong Mod Weak
CLZ PB SDMRIF SPZ DEXRIT* CMZ PHN
DTBA
Faucette S et al. Drug Metab Dispos 2004
0
50
100
150
200
250
300
350
400
450
CT
L
CM
Z
CL
Z
DE
X
DT
BA
MT
X
PA
X
PB
PH
N
PR
OB
RIF
RIT
SD
M
SP
Z
TAO
CY
P2
B6
Ac
tiv
ity
(p
mo
l/m
in/m
g p
rote
in)
2 uM (or 50 uM for PB)
10 uM (or 150 uM for PB)
20 uM (or 250 uM PB)
0
2000
4000
6000
8000
10000
12000
14000
CT
L
CM
Z
CL
Z
DE
X
DT
BA
MT
X
PA
X
PB
PH
N
PR
OB
RIF
RIT
SD
M
SP
Z
TAO
CY
P3A
4 A
ctiv
ity
(pm
ol/
min
/mg
pro
tein
)
Co-regulation of CYP2C9 and CYP3A4 by Avasimibe
HH 1 (CYP2C9)
0
20
40
60
80
100
120
0.001 0.01 0.1 1 10 100
Log Avasimibe Concentration
CY
P2C
9 A
ctiv
ity
(% o
f M
axim
um
) PredictedObserved
HH 1 (CYP3A4)
0
20
40
60
80
100
120
0.001 0.01 0.1 1 10 100
Log (Avasimibe Concentration)
CY
P3A
4 A
ctiv
ity
(% o
f M
axim
um
) Predicted
Observed
HH 2 (CYP2C9)
0
20
40
60
80
100
120
0.001 0.01 0.1 1 10 100
Log Avasimibe Concentration
CY
P2C
9 A
ctiv
ity
(% o
f M
axim
um
) PredictedObserved
HH 2 (CYP3A4)
0
20
40
60
80
100
120
0.001 0.01 0.1 1 10 100
Log (Avasimibe Concentration)
CY
P3A
4 A
ctiv
ity
(% o
f M
axim
um
) Predicted
Observed
Induction of CYP2C8 RNA
0
1
2
3
4
5
CMZ 2 CMZ 10 CMZ 50 PHY 50 CLZ 10 RIF 10 PB 1000 CITCO 1
Fo
ld o
ver
con
tro
lInduction of CYP2C9 RNA
0.0
0.5
1.0
1.5
2.0
2.5
3.0
CMZ 2 CMZ 10 CMZ 50 PHY 50 CLZ 10 RIF 10 PB 1000 CITCO1
Fo
ld o
ver
con
tro
l
Induction of CYP2C19 RNA
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
CMZ 2 CMZ 10 CMZ 50 PHY 50 CLZ 10 RIF 10 PB 1000 CITCO1
Fo
ld o
ve
r c
on
tro
l
Mechanism-based Screening Strategy
Goal is to screen for efficacious activators of AhR, PXR, and CAR
Propose screening protocol using sensitive endpoint for each nuclear receptor
Potent activators of individual NR’s will induce a number of target genes, but differentially:– Potent hPXR activators will induce CYP3A4, CYP2B6,
CYP2C8/9/19, UGT1A1, MDR1 etc., but CYP3A4 is the most sensitive.
– Potent hCAR activators will induce CYP2B6, CYP2C8/9/19, UGT1A1 etc., but CYP2B6 is the most sensitive.
– Potent AhR agonists will induce CYP1A2, UGT1A1, GST’s etc., but CYP1A2 is the most sensitive.
In Vitro Protocol for Enzyme Induction
Cultured HepatocytesCultured Hepatocytes
Treat with NME at 3-4 Treat with NME at 3-4 conc. for 1-3 daysconc. for 1-3 days
Include Positive Include Positive Controls:Controls:3-MC/BNF3-MC/BNF
Phenobarbital/PhenytoinPhenobarbital/PhenytoinRifampinRifampin
mRNA ContentRT/PCR (TaqMan)
Protein ContentWestern Immunoblotting
Enzyme ActivityLC-MS/MS assayHPLC assayRadiometric assay
Major CYP target gene for each nuclear receptor:Major CYP target gene for each nuclear receptor:CYP1A2 CYP1A2 ((AhRAhR)), 2B6 , 2B6 (CAR(CAR)),, 3A4 3A4 ((PXRPXR))
-4000
-2000
0
2000
4000
6000
8000
Probe
necid
Rifam
pin
Ritona
vir
Sulfin
pyra
zone
Carba
mez
apin
e
Clotri
maz
ole
Dexam
etha
sone
Pheny
toin
Treatment Group
No
rmal
ized
sp
ecif
ic a
ctiv
ity
(pm
ol/m
in/m
g p
rote
in)
2 uM
10 uM
20 uM
CYP3A4 Induction in Human Hepatocytes
0
5
10
15
20
25
Probenecid Rifampin Ritonavir Sulfinpyrazone
Treatment Group
No
rmal
ized
fo
ld in
du
ctio
no
ver
con
tro
l
2 uM
10 uM
Effects of Inducers on CYP3A4mRNA Expression
Important Factors to Consider
Inter-donor differences in control/basal activity
Relevant concentration range (plasma vs. tissue)
Appropriate choice and concentration of positive controls
Major species differences exist (e.g., RIF vs. PCN)
Expression of data and relevant endpoints
Exposure time important (# days)
Solvent effects on CYP450 expression and activity
Summary of Key Points
Our mechanistic understanding of enzyme induction in human liver has increased markedly in the past decade.
Most inducible human P450’s, UGT’s and transporters involved in DDI’s are regulated by a few receptors (i.e., PXR, AhR and CAR).
Screening for potential inducers during drug development can be achieved using a single selective and sensitive target gene for each NR.
Activity data from in vitro induction studies for a NME should be:
– normalized to a negative control – compared to an ‘appropriate’ positive control– considered significant when they are 40% of the positive
control complemented with protein or mRNA data