webinar: application of inside-out vesicles for accessing ... · bd biosciences february 9, 2011...

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

6

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

7

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

10

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

11

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

12

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

13

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

14

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)

15

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

18

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




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)


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





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


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


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.

28

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

webinar: application of inside-out vesicles for accessing ... · bd biosciences february 9, 2011...

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