Webinar: Application of Inside-Out Vesicles for Accessing ABC … · 2014-03-11 · 2 BD Gentest™...
Transcript of Webinar: Application of Inside-Out Vesicles for Accessing ABC … · 2014-03-11 · 2 BD Gentest™...
BD Biosciences
February 9, 2011
Application of Inside-Out Vesicles for Accessing ABC Transporter Inhibition and Direct Transport
Na Li, PhD
2
BD Gentest™ Transporter Seminar Series
• Today’s seminar is the one of a series of transporter seminars that BD will present through 2010 and 2011.
• The upcoming titles and dates are as follows:– Drug Transport Efflux and Uptake Assays Using Plated and
Suspension Hepatocytes: May 5, 2011
– Application of Transportocytes for Identifying Inhibitors andSubstrates of SLC Transporters: October 5, 2011
3
Today’s Topics
• Overview of the important role of ABC transporters in drug ADME and toxicity.
• Overview of in vitro ABC transporter models
• Application of vesicle uptake assay in characterizing transporter substrates and inhibitors – Assay procedure
– Application in characterizing drug interactions with major ABC transporters
4
Transporters Across Human Tissues
Kitamura S, et al. Naunyn Schmiedebergs Arch Pharmacol. 377(4-6):617-28 (2008).
5
Transporters in Drug Pharmacokinetics and Toxicity
• Systemic exposure: Oral bioavailability and Organ Disposition– Drug absorption in small intestine (BCRP, MDR1/P-gp)
(Topotecan, sulphasalazine)
• SLC transporter involved DDI– OATP1B1: increase in statin AUC when co-administrated
with cyclosporine
• MDR1/P-gp involved DDI– Digoxin clearance reduced when co-administrated with Quinidine,
Ritonavir, and other P-gp inhibitors
• Inhibition of MRP2/BSEP induced toxicity– Cholestasis
– Hyperbilirubinemia
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Met
abol
ic s
tabi
lity
Low
Moderate
High
Con
fide
nce
in p
redi
ctio
n
Extensive P450 or Phase II enzyme mediated drug metabolism, e.g. High drug clearance Rational compound structure optimization is needed
Optimal P450 or Phase II enzyme mediated drug metabolism, e.g. moderate drug clearance DDI potential, metabolites, interspecies scaling and IVIVC are assessable
Metabolic stabilityunknown routes of elimination (all transporter mediated)DDI, interspecies scaling and IVIVC are difficult to assess
Transporter-Mediated Drug Clearance –Challenge in Drug Discovery
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Selected Human Transporters
Figure from Nature Reviews: Drug Discovery (2010) 9:215-236 Recombinant transporters available from BD
8
ABC transportersSLC transporters
KidneyMATE2-K (SLC47A2)
Kidney, liver, skeletal muscle
MATE1 (SLC47A1)
Kidney, lungPEPT2 (SLC15A2)
Intestine, kidneyPEPT1 (SLC15A1)
Liver, intestineOCT1 (SLC22A1)
LiverMDR3 (ABCB4)LiverOATP2B1/OATP-B (SLCO2B1)
Kidney, liver, MRP4 (ABCC4)BrainOATP1A2/OATP-A (SLCO1A2)
Liver, intestineMRP3 (ABCC3)Kidney, brainOCT2 (SLC22A2)
Liver, kidney, intestineMRP2/cMOAT (ABCC2)Kidney, liver, brainOAT3 (SLC22A8)
LiverBSEP (ABCB11)Kidney, placentaOAT1 (SLC22A6)
Intestine, liver, kidney, brain, placenta, breast
BCRP/MXR (ABCG2)LiverOATP1B3/OATP-8 (SLCO1B3)
Intestine, Kidney, liver, brainMDR1/P-gp (ABCB1)LiverOATP1B1/OATP-C, OATP2 (SLCO1B1)
Organ/cellsTransporters/aliasTissue Distribution
Transporters/alias
Clinical Drug drug interaction white paper, 2006, FDA
Membrane Transporters in Drug Development, ITC, Nature Review/Drug Discovery,March (vol 9):215-236 (2010).
EMA, 2010
Drug Transporters of Emerging Clinical Importance in the Absorption and Disposition of Drugs
FDA 06’
ITC 10’
ITC 10’
EMA 10’
EMA 10’
9
Overview of In Vitro Transporter Models
SLC transporters: • Expressed in Xenopus oocyte system (Transportocytes)
– OATP, OCT, OAT, and NTCP – Uptake assays for both radiolabeled and non-radiolabeled compounds
• SLC transporters overexpressed in mammalian cell lines• Hepatocyte suspension assay using oil-filtration method
ABC transporters:• Inside-out vesicles:
– MRP, BCRP, and BSEP– Uptake assays for both radiolabeled and non-radiolabeled compounds
• Membranes: – P-gp, MRP, and BCRP – ATPase assay
• Polarized cell line expressing human P-gp, MRP2 or BCRP• Sandwich cultured hepatocytes-ABC transporter on canalicular membrane
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Monolayer Efflux Studies In Polarized Cell Monolayers
Apical Side
Basolateral Side
Apical Side
Basolateral Side
Compound Measure
CompoundA B B A
Apical Side = DonorBasolateral Side = Receiver
Apical Side = ReceiverBasolateral Side = Donor
Measure
PP--gpgp, MRP2, BCRP , MRP2, BCRP overexpressedoverexpressed in LLPK, or MDCK cellsin LLPK, or MDCK cells
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Sandwich Cultured Hepatocytes── Hepatic Uptake and Biliary Excretion
Advantage:Repolarization of hepatocytes
Form intact bile canaliculi, mimic in vivo hepatobiliary network
Quantitative estimation of hepatic uptake clearance and biliary excretion
Disadvantage:Alteration of expression level of various of basal and apical transporters compared to in vivo rat livers.
Fresh Hepatocytes
CryoHepatocytes
p450
Bile
GSH/glucuronide
p450
Tight Junction
Collagen I
BD Matrigelmatrix
Hepatocytes
BD BiocoatTM Collage I plate
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ATP
ADP + PiDrug
Vesicles Uptake Assay
PGP
ATP ADP + Pi • BD Gentest ABC transporter Vesicles
• Direct Assay: Measurement of transporter-mediated uptake of drugs into vesicles (Substrates and inhibitors)
• Can be high throughput: using 96-well plateand cell harvester / vacuum manifold
• Assays developed for both radiolabeled and non-radiolabeled compounds
• BD Gentest™ ABC transporter membrane
• Indirect Assay: measures the ability of a drug-stimulated ATP hydrolysis (substrates and inhibitors)
• Rapid, High-throughput • Colorimetric assay (any compound)• No extractions/separations/transfers
ATPase Assay
ABC Transporter Assays
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BD Gentest ABC TransporterMembranes / Vesicles
Membranes for ATPase Assay• Human MDR1 (P-gp)• Mouse Mdr1a/1b• Rat Mdr1a/1b• Cyno Mdr1• Rhesus Mdr1• Beagle Dog Mdr1
• Human MRP1• Human MRP2• Human MRP3• Rat Mrp1• Rat Mrp2
• Human BCRP (Arg482)• Mouse Bcrp• Rat Bcrp
Inside-out Vesicles for Uptake Assay• Human MRP1 • Human MRP2• Human MRP3• Rat Mrp1 • Rat Mrp2
• Human BSEP• Rat Bsep
• Human BCRP (Arg482)• Rat Bcrp• Mouse Bcrp
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Transporter Assay Kits
NEW ProductsNEW Products
• ATPase Kit (cat. no 459006)– One kit supports all BD ABC transporter membrane ATPase assays– 5 plates of 96-well plate assay– Probe substrates for P-gp, MRP1, MRP2, MRP3 and BCRP.– Colorimetric reagents in single use aliquot– 2 Year shelf-life when store at -20oC– Reagents only
• MRP/BCRP Vesicle Kit (cat. no. 459010)– One kit supports all BD MRP and BCRP transporter vesicle assays– 200 assays – Probe substrate for MRP1, MRP2, MRP3 and BCRP – Fluorescent substrate (CDCF) for MRP2 and MRP3– 2 Year shelf-life – Reagents only
• BSEP Vesicle Kit (cat. no 459011)– Supports BSEP transporter vesicle assays– 200 assays– Probe substrate for BSEP– 2 Year shelf-life– Reagents only
• 10 X Wash Buffer for MRP/BCRP Vesicle Assay (cat. no 450600)• 10 X Wash Buffer for BSEP Vesicle Assay (cat. no 450601)
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Vesicle Uptake Assays
16
Vesicle Uptake Assay
Vacuum
Filter(facing down)
Well
ATP
ADP + Pi
Drug
Inside-out Vesicles Cell Harvester
Filter Plate
Equipment and Materials
Microplate Scintillation Counter
Vacuum Manifold
Fluorescence Reader LC-MS/MS
17
Using Radio-labeled Compounds in Vesicle Uptake Assays
• Equipment– Vacuum manifold/cell harvester and scintillation counter (if 96-well glass
fiber filter plate is used, MicroBeta scintillation counter, Perkin Elmer is needed)
• Advantages– Extraction step in sample preparation is not required– Recovery is not an issue– Fast
• Disadvantages– Radiolabeled compounds are not always available– Waste removal and radiation license requirements associated
with radiolabeled compound– Expensive
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Overview of Assay Procedure – Example of BSEP IC50 Study
Uptake activity with ATP
Uptake activity with AMPATP-dependent uptake activity = Uptake activity with ATP (pmol/min/mg) - Uptake activity with AMP (pmol/min/mg)Signal to Noise Ratio= Uptake activity with ATP (pmol/min/mg)___
Uptake activity with AMP (pmol/min/mg)
IC50 Calculation:
19
Characterization of MRP2 mediated Uptake Leukotriene C4 (LTC4)
Protein-dependent
0.0
0.1
0.2
0.3
0.4
0.5
0 20 40 60 80 100 120
Protein concentration (μg)
LTC
4 U
ptak
e in
hM
RP2
Ves
icle
(p
mol
/min
)
A
0
10
20
30
40
0 1 2 3 4 5 6 7Time (min)
LTC
4 U
ptak
e A
ctiv
ity in
hM
RP2
Ves
icle
(pm
ol/m
g)
B Time-dependentLTC4 Concentration (μM)
0 1 2 3
0
20
40
60
80
100
120
ATP-
depe
nden
t Upt
ake
Activ
ity o
f LTC
4 (p
mol
/mg/
min
)
LTC4 uptake in hMRP2 VesicleC
Km: 5.9±0.5 μM
Vmax: 279.4±17.1 (pmol/min/mg)
Incubation time: 4min
Protein concentration: 50 μ g
with ATP without ATP
20
Km: 14.2±1.9 μM
Vmax: 506.0±25.3 (pmol/min/mg)
Incubation time: 3min
Protein concentration: 50 μ g
E3S Uptake in hBCRP Vesicle
E3S concentration (μM)0 20 40 60
0
100
200
300
400
500
ATP-
depe
nden
t Upt
ake
Activ
ity o
f E3
S (p
mol
/mg/
min
)
CProtein-dependent
0.0
1.0
2.0
3.0
4.0
5.0
6.0
0 20 40 60 80 100 120Protein Concentration (μg)
E3S
Upt
ake
Act
ivity
in h
BC
RP
(pm
ol/m
in)
A
Time-dependent
0
50
100
150
200
250
0 1 2 3 4 5 6 7Time (min)
E3S
Upt
ake
Activ
ity in
hBC
RP
(pm
ol/m
g)
B
Characterization of BCRP mediated Uptake (Estrone-3-Sulfate)
with ATP without ATP
21
Characterization of BSEP mediated Uptake (Taurocholate)
Time-dependent
0153045
607590
105
0 10 20 30 40 50Time (min)
TCA
Upt
ake
Act
ivity
in h
BSE
PVe
sicl
e (p
mol
/mg)
B
Protein-dependent
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0 20 40 60 80 100 120Protein concentration (μg)
TCA
Upt
ake
Act
ivity
in h
BSE
PVe
sicl
e (p
mol
/min
)
A
TCA Concentration (μM)0 10 20 30 40 50 60 70
0
5
10
15
20
ATP-
depe
nden
t Upt
ake
Activ
ity o
f TC
A
(pm
ol/m
g/m
in)
TCA uptake in hBSEP VesicleC
Km: 8.4±0.6 μM
Vmax: 20.1±0.5 (pmol/min/mg)
Incubation time: 10min
Protein concentration: 50 μ g
with ATP without ATP
22
Summary of ABC Transporter Vesicle Kinetic Data
Noe J et al., Gastroenterology 2002, 123, 1659-1666; Byrne JA et al., Gastroenterology 2002, 123, 1649-1658
8.0/ 4.38.4±0.6TCAhBSEP
Karlsson JE, et al, DMD, 20101114.2±1.9E3ShBCRP
Cui Y et al., Mol. Pharm., 55, 929, 1999; Bakos E et al., Mol. Pharm., 57, 760, 2000
1.0/1.4 5.9±0.5LTC4hMRP2
LiteratureReference Km (μM)
Km (μM)Day 1SubstrateTransporter
23
IC50 Determination of MRP2 Transporter Modulators
IC50 of hMRP2 inhibitor (LTC4)
% o
f Rem
aini
ng L
TC4
Upt
ake
Act
ivity
in
hM
RP
2 V
esic
les
Concentration of Inhibitor (μM)0.1 1 10 100 1000 10000
-20
0
20
40
60
80
100
120
MK571BenzbromaroneTerfenadineIndomethacin
A.
281.5301262Indomethacin
41.642.240.9Terfenadine
7.88.37.3Benzbromarone
9.29.58.9MK571
Average IC50 (uM)IC50 (uM) Day 2IC50 (uM) Day 1Test compounds
24
IC50 Determination of BCRP Transporter Modulators
IC50 of hBCRP inhibitor (E3S)
Concentration of Inhibitor (μM)0.0001 0.001 0.01 0.1 1 10 100
0
20
40
60
80
100
120
SulfasalazineNobomycinFumitremorgin CKO143
0.00.0240.026K0143
0.10.10.1FTIC
0.60.60.5Nobomycin
0.70.70.7Sulfasalazine
Average IC50 (uM)IC50 (uM) Day 2IC50 (uM) Day 1Test compounds
25
IC50 Determination of BSEP Transporter Modulators
IC50 of hBSEP Uptake (TCA)
Concentration of Inhibitor (μM)0.1 1 10 100 1000
-20
0
20
40
60
80
100
120
Troglitazone
Cyclosporin AGlibenclamide
0.620.620.62Cyclosporin A
3.54.592.4Glibenclamide
4.454.064.84Troglitazone
Average IC50 (uM)IC50 (uM) Day 2IC50 (uM) Day 1Test compounds
26
Non-Specific Binding of Probe Substrate on GF/B filter plate
75uptake buffer
15015010vesicle (5mg/ml)
37575075050Solution A
(substrate Mix)
Blank(lot 2)(lot 1)volume (ul)
Reaction solution prep
Raw date of Microbeta Counter
52535351H
578522121231051G
774818391123154F
89621780996553E
671731447859D
671878486960C
712122438761B
57565757A
4321
Blank
(substrate only)
27
Comparison of 1%BSA and 100uM TCA ─── blocking non-specific binding of TCA on GF/B filter plate
comparison of blocking reagents
0.00
1000.00
2000.00
3000.00
4000.00
5000.00
6000.00
7000.00
8000.00
no blocking 1% BSA blocking 100uM TCA blocking
Raw
dat
a ATP-ATPblank
S/N 2.4
S/N 4.6
S/N 2.8
• 1% BSA is more sufficient to block the non-specific binding of TCA on GF/B filter plate, comparing with 100uM cold TCA substrate
• Blocking with 1% BSA significantly enhanced the S/N ratio of failing lot 40193.
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BD Gentest Vesicle Assay Kit
MRP1/MRP2 Probe Substrate100ul 100 μM LTC4
MRP2/MRP3 Probe Substrate100ul 1mM CDCF
BCRP Probe Substrate100ul 1mM Estrone-3-sulfate
MRP2/MRP3 Probe Substrate50ul 20mM Estradiol-17β-glucuronide
Cofactor100ul 300mM GSH
Control500ul 200mM AMP
Energy 500ul 200mM MgATP
Wash Buffer50ml 10x MOPs Wash buffer
Uptake Buffer20ml MOPs Uptake buffer
DescriptionComponent
200 assays
Shelf-life: 2 years
Blocking Buffer1.0ml 5% BSA (50X)
BSEP Probe Substrate100ul 1mM taurocholic acid
Control500ul 200mM AMP
Energy500ul 200mM MgATP
Wash Buffer60ml 10x Hepes Wash buffer
Uptake Buffer20ml Hepes Uptake buffer
DescriptionComponent
MRP/BCRP Vesicle Assay Kit
BSEP Vesicle Assay Kit
29
Vesicle Assay Kit Freeze-thaw Stability
The loss of ATP-dependent probe substrate uptake activity after 6 times freeze-thaw was less than 20% across the panel of tested ABC transporter vesicles.
LTC4/hMRP2
CDCF/hMRP2
E3S/hBCRP
E17BG/hMRP3
TCA/hBSEP
ATP
-dep
ende
nt U
ptak
e A
ctiv
ity (p
mol
/mg/
min
)
0
10
20
30
40
501 X 6 X
30
Consistent Performance of Vesicle Assay Kit (Lot to Lot Variance)
LTC
4 up
take
in h
MR
P2 V
esic
le
MRP/BCRP Vesicle Kit (hMRP2)
0
2
4
6
8
10
12
Lot 1 lot 2 lot 3
(pm
ol/m
g/m
in)
12.0%0.8%S/NS-N
S/N 8.2 S/N 9.4 S/N 10.4
A
MRP/BCRP Vesicle Kit (hBCRP)
0
10
20
30
40
50
60
Lot 1 Lot 2 Lot 3
E3S
upta
ke in
hB
CR
PVe
sicl
e (p
mol
/mg/
min
)
ATP
AMP
9.6%3.0%S/NS-N
S/N 4.8 S/N 4.0 S/N 4.2
B
BSEP Vesicle Kit (hBSEP)
0.0
0.5
1.0
1.5
2.0
2.5
Lot 1 Lot 2 Lot 3
TCA
Upt
ake
in h
BSE
PVe
sicl
e (p
mol
/mg/
min
)
13.6%4.6%S/NS-N
S/N 6.5 S/N 6.3 S/N 5.0
C.
• Lot-to-lot variance in ATP-dependent uptake activity (S-N) is less than 5%
• Lot-to-lot variance in S/N ratio is less than 15%
31
Summary
• Awareness is increasing on the importance of drug transporters in drug pharmacokinetics, safety and efficacy profiles.
• Vesicle uptake assays are direct assays to determine transporter substrates or inhibitors.
• Vesicle assay kit is a more robust and convenient tool to characterize drug interactions with MRP, BCRP and BSEP transporters.
32
Summary of BD BiosciencesIn Vitro Transporter Models
• ABC Transporters– Inside-out vesicles
• Uptake and inhibitory assays • MRP/BCRP vesicle kit (459010); BSEP vesicle kit (459011)• 10X wash buffer for MRP/BCRP vesicle assay (450600)• 10X wash buffer for BSEP vesicle assay (450601)
– Membranes• ATPase assay• ATPase assay kit (459006)
– Fresh Hepatocytes and CryoHepatocytes– MDR1-LLPK cells
• SLC Transporters – Expressed in Xenopus oocyte system (transportocytes)
• Uptake and inhibitory assays– Hepatocyte suspension assay using oil-filtration method
• Uptake and inhibitory assays
33
Questions?
Contact information:Na Li, PhDe-mail: [email protected]
Technical Support:tel: 877.232.8995e-mail: [email protected]/webinarsFor research use only. Not intended for use in diagnostic or therapeutic procedures. BD, BD Logo, and all other trademarks are property of Becton, Dickinson and Company. ©2010 BD