na li, phd · 2014-03-11 · polarized cells line (mdr1-llpk cell line and mdr1-mdckii cell line)...
TRANSCRIPT
BD Biosciences
December 8, 2010
Application of the Colorimetric ATPase Assay for Accessing ABC Transporter Inhibition and StimulationNa Li, PhD
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BD Gentest™ Transporter Seminar Series
• Today’s seminar is the first in a series of transporter seminars that BD will present through 2010 and 2011.
• The upcoming titles and dates are as follows:– Application of inside-out vesicles for accessing ABC transporter
inhibition and direct transport: February 9, 2011
– Application of Transportocytes for identifying inhibitors and substrates of SLC transporters: April 7, 2011
– Drug transport efflux and uptake assays using plated andsuspension hepatocytes: May 5, 2011
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Today’s Topics
• Overview of the important role of transporters in drug ADMET
• Comparing in vitro transporter models for studying P-gp interaction
• Application of Colorimetric ATPase assays in characterizing drug interaction with ABC transporters– Assay procedure
– Characterization and validation of BD Gentest ATPase assay kit
– Application in characterizing drug interactions with ABC transporters, P-gp, MRP2 and BCRP.
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Transporters Across Human Tissues
Kitamura S, et al. Naunyn Schmiedebergs Arch Pharmacol. 377(4-6):617-28 (2008).
Phase I, II drug metabolism
Phase III transporter-mediated drug elimination
Bioavailability
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Transporters in Blood Brain Barrier (BBB)
Mayer, U., et al. British J. Pharmacol. 119:1038-1044 (1996).
Plasma Brain
Schinkel, A.H., et al.Advanced Drug Delivery Reviews. 36:179-194 (1999).
ABC transporter: P-gp, BCRP
SLC transporter: OATP1A2, OAT3 and MCT1
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Transporter in Small Intestine
• Systemic exposure: Oral bioavailability and Organ Disposition
– Drug absorption in small intestine• MDR1/P-gp (Paclitaxel)• BCRO (Topotecan)
ABC transporter: P-gp, BCRP, MRP2
SLC transporter: OATP, PEPT1, ASBT, MCT1
Mdr1a/1b-/- mice:Pre-dose elacridar(GF120918) (50 mg/kg)
Oral dose of Topotecan(1 mg/Kg)
A cohort of 8 patients:A single oral dose of topotecan (1 mg/m2) with or without a single does of oral elacridar (1000 mg)
6 fold increase in the plasma concentration
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Hepatobiliary Transporter System
Blood
Organic anions/ cation
MRP3
ABCG2(BCRP)
ABCG5ABCG8
Cholesterol
MDR3
MRP2
BSEP(PFIC2/3)
MDR1
Na+
Bile salts
OATPsOATP-AOATP-BOATP-8
NTCP
OCT1
OAT2 Conjugates GSH, glucuronide
Lipid Cation
phospholipidsSulfoconjugates
glucuronidesMRP4 MRP5 MRP6
SulfoconjugatesBile salts cGMP
Organic anions
CNT1/2
Bile
Sinusoidal membrane
• 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
Central Vein
Portal Vein
Hepatic artery
Bile duct
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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’
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Overview of in vitro Transporter Models
SLC transporters: – Transportocytes: SLC transporter expressed in Xenopus oocyte
• OATP, OCT, OAT, and NTCP • Uptake assays for both radiolabeled and non-radiolabeled compounds
– SLC transporters overexpressed in mammalian cell lines– Hepatocyte suspension uptake assay: 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
– Monolayer Efflux Assay: Polarized cell line expressing humanP-gp, MRP2 or BCRP
– Sandwich cultured hepatocytes: Hepatic uptakevia SLC transporter on the sinusoidal membrane andbiliary excretion via ABC transporter on the canalicular membrane.
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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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• Monolayer Efflux Studies: P-gp Overexpressedpolarized cells line (MDR1-LLPK cell line and MDR1-MDCKII cell line)
• Colorimetric ATPase assay
• Calcein Inhibition Assay– Calcein-AM: permeable compound, non fluorescent,
P-gp substrate
– Celcein: Calcein-AM hydrolysis product, non permeable compound, fluorescent
Major in vitro Models to Study P-gp
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Apical Side
Basolateral Side
Apical Side
Basolateral Side
Compound Measure
CompoundA B B A
Apical Side = DonorBasolateral Side = Receiver
Apical Side = ReceiverBasolateral Side = Donor
Monolayer Efflux Studies In Polarized Cell Monolayers
Measure
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Colorimetric ATPase Assay
Drug + ABC transporter membrane
Stop Reaction by 10%SDS
colorimetric reagents
Absorbance read at 800 nm
Read-out on SpectrometerColorimetric Assay
ExtracellularExtracellular
CytoplasmCytoplasm
PGP
ATP ADP +
Substrate
Pi
Blue complex
• 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
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Permeability and concentration should be taken into consideration.• Higher than 300 nm/s:
– No for monolayer transport (false negative)– Yes for ATPase
• Lower than 20nm/s:– Yes for transport– Y/N for ATPase. Concentration at the site is critical.
• Some substrates could interfere with the ATPase activity at highconcentration range
• Reference: – Polli, J., et al., JPET 299:620-628 (2001).– Shirasaka, Y., et al., Biol. Pharm. Bull. 29:2465-2471 (2006).
Correlation Between ATPase Assay vsTransport Assay
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BD Gentest 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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Colorimetric ATPase Assays
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Using Radiolabeled Compounds in Vesicle Uptake Assays
• Equipment– Vacuum manifold/cell harvester and scintillation counter
(if 96-well glass fiber filter plate is used, MicroBeta scintillationcounter, 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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hBCRP (Arg482) membrane: 20 g
Substrate: 1 M PhIP
ATP: 4mM
NaOV: 0.4mM
Incubation time: 10min
STD Curve
Signal
Noise
Drug-stimulated activity
NaOV Sensitive activity
The noise can vary a lot.
When the noise is zero or negative, the S/N ratio is not applicable, shown as N/A in the following presentation.
How to Set Up an ATPase Assay
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1. Phosphate free: Be sure all reagents, solvent, glass vials, tubes and reservoirs are free of phosphate.
2. No bubbles: Bubbles will interfere with absorbance.
3. Change pipette tips frequently to avoid cross-contamination.
4. Solvent tolerance is ≤ 2%.
Keys to Success (ATPase Assay)
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Compound Categories
N: negative result in the assay
Y: positive result in the assay
YYY
Monolayer Efflux Assay P-gp ATPase Assay Calcein Inhibition Assay
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Correlation of P-gp Associated ATPase Activity with Efflux Ratio Determined in MDCK-MDR1/P-gp Cell-based Assay
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Correlation of P-gp Associated ATPase Activity with Efflux Ratio Determined in MDCK-MDR1/P-gp Cell-based Assay
Correlation of ATPase Assay Result with Monolayer Efflux Assay
S/N Ratio (BD ATPase Assay Kit)
0.1 1 10 100
Effl
ux R
atio
(MD
CK
-MD
R1
Mon
olay
er E
fflux
Ass
ay)
0.1
1
10
100
1000
False negative
False Positive
Positive
Negative
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Correlation of MDR1/P-gp ATPase Km Values with Efflux Ratio in MDCK-MDR1 Cell Assay
* The binding affinities (αKa) of saquinavir and ritonavir were calculated on the basis of a nonessential enzyme activation model (Cindy XQ, et al, Molecular Pharmaceutics, 2006)
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Substrate Stimulated BCRP ATPase Assay
Vmax=46.3 ±2.0 (nmol/mg/min) Km = 3.8 ±0.7 µM Incubation Time: 10 min Protein: 20 µg
PhIP
[PhIP] (µM)
0 20 40 60 80 100 120
Rat
e (n
mol
/mg/
min
)
0
10
20
30
40
50A Sulfasalazine
[Sulfasalazine] (µM)0 20 40 60 80 100 120
Rat
e (n
mol
/mg/
min
)
2
4
6
8
10
12
Vmax=10.6 ±0.6 (nmol/mg/min) Km = 1.0 ±0.3 µM Incubation Time: 20 min Protein: 20 µg
B
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Substrate Stimulated MRP2 ATPase Assay
Vmax=9.2 ±0.4 (nmol/mg/min) Km = 24.6 ±3.9 µM Incubation Time: 40 min Protein: 20 µg
Vmax=10.3 ±2.1 (nmol/mg/min) Km = 0.9 ±0.4 mMIncubation Time: 40 min Protein: 20 µg
Probenecid
[Probenecid] (mM)0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
Rat
e (n
mol
/mg/
min
)
0
2
4
6
8
10Sulfasalazine
Rat
e (n
mol
/mg/
min
)
123456789
[Sulfasalazine] (µM)0 100 200 300
BA
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BD Gentest ATPase Assay Kit
0.1 mLMRP3/Mrp3 positive control 25 mM Benzbromarone
0.2 mLGSH, MRP1/Mrp1 positive control 1 M L-Glutathione
0.2 mLMRP1/Mrp1 positive control 1 M N-Ethylmaleimide
0.2 mLMRP2/Mrp2 positive control 100 mM Probenecid
0.4 mLMDR1/Mdr1 positive control 1 mM Verapamil
0.2 mLBCRP/Bcrp positive control 1 mM Sulfasalazine
5 x 2 gAscorbic acidReducing Agent
5 x 3 mL30 mM Zinc AcetateColor Solution B
5 x 3 mL70 mM Ammonium MolybdateColor Solution A
25 mL10% SDS solutionAssay Stop Solution
2 x 1.0 mLNaOV, ATPase Inhibitor10 mM Sodium Orthovanadate
2 x 1.75 mLATP50 mM ATP
1.7 mLStandard Curve10 mM Phosphate Standard
2 x 30 mLTris-Mes Buffer (pH 6.8)Assay Buffer
QuantityDescriptionName
Storage Temp: -20oC
Shelf-life: 2 years
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0.0
10.0
20.0
30.0
40.0
50.0
60.0
Vana
date
-Sen
sitiv
e Su
bstr
ate
Stim
ulat
ed
ATP
ase
Act
ivity
(nm
ol/m
g/m
in)
2x4x6x8x
50 μMBenzbromarone
1mM Probenecid10mM NEM GSH10 μM PhIP20 μM Verapamil
Human MRP3Human MRP2Human MRP1Human BCRPHuman P-gp
The loss of probe substrate stimulated ATPase activity after 8 times freeze-thaw was less than 20% across the panel of tested ABC transporter membrane.
Multiple Freeze-thaw Stability of ATPase Assay Kit
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Consistent Performance of ATPase Kit (Lot to Lot Variance)
S-N: 9.9% S/N 24.8%
S-N: 10.6% S/N 16.4%• Lot-to-lot variance in vanadate sensitive
ATPase activity is less than 20%
• Lot-to-lot variance in S/N ratio is less than 30%
Lot to Lot Variance of ATPase Kit (hMRP2)
0
2
4
6
8
10
12
14
16
18
Lot 1 Lot 2 Lot 3Subs
trat
e-st
imul
ated
ATP
ase
Act
ivity
(n
mol
/mg/
min
) S/N 19 S/N 14S/N 12
Lot to Lot Variance of ATPase Assay Kit (hBCRP)
0102030405060708090
Lot 1 Lot 2 Lot 3
Subs
trat
e-st
imul
ated
ATP
ase
Act
ivity
(nm
ol/m
g/m
in)
S/N 5.6 S/N 5.2S/N 7.1
Lot to Lot Variance of ATPase Kit (human P-gp)
0
5
10
15
20
Lot 1 Lot 2 Lot 3Subs
trat
e-st
imul
ated
ATP
ase
Act
ivity
(nm
ol/m
g/m
in)
S-N: 16.6 % S/N 14.8%
S/N 17.7S/N 12.6 S/N 16.1
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Summary
• Evidence has demonstrated the clinical importance of drug transporters, e.g. drug pharmacokinetics, safety and efficacy profiles
• The BD Gentest ATPase assay kit was validated for performing the ATPase assay to characterize drug interaction with ABC transporters.
• The signal-to-noise ratio of selected P-gp substrates or non-P-gpsubstrates was consistent with literature and correlated with the efflux results determined in MDCK-MDR1 cell bi-directional assay.
• The Km of selected P-gp modulators was determined using the BD Gentest ATPase assay kit and BD Gentest ABC transporter membranes. The data were comparable with literature reports.
• Colorimetric ATPase assay is a predictive and high throughput in vitro transporter model to measure drug interaction with transporters.
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Summary of BD Biosciences in vitro Transporter Models
• ABC Transporters– Inside-out vesicles
• Uptake and inhibitory assays • MRP/BCRP vesicle kit (cat. no. 459010); BSEP vesicle kit (cat. no. 459011)• 10X wash buffer for MRP/BCRP vesicle assay (cat. no. 450600)• 10X wash buffer for BSEP vesicle assay (cat. no. 450601)
– Membranes• Screening assay or kinetics assay• ATPase assay kit (cat. no. 459006)
• SLC Transporters – Expressed in Xenopus oocyte system (transportocytes)
• Uptake and inhibitory assays– Hepatocyte suspension assay using oil-filtration method
• Uptake and inhibitory assays
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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