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: na_li@bd.com

Technical Support:tel: 877.232.8995e-mail: labware@bd.combdbiosciences.com/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