ABC Transporter Vesicle Assay: Comparison of Cold (LC-MS/MS) and Hot (Radiolabeled) Methods. Na Li, PhD. BD Biosciences Discovery Labware May 29, 2012

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1 ABC Transporter Vesicle Assay: Comparison of Cold (LC-MS/MS) and Hot (Radiolabeled) Methods Na Li, PhD BD Biosciences Discovery Labware May 29, 212

2 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 Hot (Radiolabeled) vesicle uptake assay procedure Cold vesicle uptake assay procedure Fluorescent compound (Spectrophotometer) Non-radiolabeled compound (LC-MS/MS) 2

3 Transporters Across Human Tissues Kitamura S, et al. Naunyn Schmiedebergs Arch Pharmacol. 377(4-6): (28). 3

4 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 4

5 Transporter-Mediated Drug Clearance Challenge in Drug Discovery Low Extensive P45 or Phase II enzyme mediated drug metabolism, e.g. High drug clearance Rational compound structure optimization is needed Confidence in prediction Metabolic stability Moderate High Optimal P45 or Phase II enzyme mediated drug metabolism, e.g. moderate drug clearance DDI potential, metabolites, interspecies scaling and IVIVC are assessable Metabolic stability unknown routes of elimination (all transporter mediated) DDI, interspecies scaling and IVIVC are difficult to assess 5

6 Selected Human Transporters Figure from Nature Reviews: Drug Discovery (21) 9: Recombinant transporters available from BD

7 Drug Transporters of Emerging Clinical Importance in the Absorption and Disposition of Drugs SLC transporters Transporters/alias OATP1B1/OATP-C, OATP2 (SLCO1B1) OATP1B3/OATP-8 (SLCO1B3) OAT1 (SLC22A6) OAT3 (SLC22A8) OCT2 (SLC22A2) OATP1A2/OATP-A (SLCO1A2) OATP2B1/OATP-B (SLCO2B1) OCT1 (SLC22A1) PEPT1 (SLC15A1) PEPT2 (SLC15A2) MATE1 (SLC47A1) MATE2-K (SLC47A2) ITC 1 Tissue Distribution Liver Liver Kidney, placenta Kidney, liver, brain Kidney, brain Brain Liver Liver, intestine Intestine, kidney Kidney, lung Kidney, liver, skeletal muscle Kidney EMA 1 ABC transporters Transporters/alias MDR1/P-gp (ABCB1) BCRP/MXR (ABCG2) BSEP (ABCB11) MRP2/cMOAT (ABCC2) MRP3 (ABCC3) MRP4 (ABCC4) MDR3 (ABCB4) FDA 6 Organ/cells Intestine, Kidney, liver, brain Intestine, liver, kidney, brain, placenta, breast Liver Liver, kidney, intestine Liver, intestine Kidney, liver, Liver ITC 1 EMA 1 Clinical Drug drug interaction white paper, 26, FDA Membrane Transporters in Drug Development, ITC, Nature Review/Drug Discovery, March (vol 9): (21). EMA, 21 7

8 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 Plateable transporter qualified cryopreserved human hepatocytes 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 Plateable transporter qualified cryopreserved human hepatocytes Sandwich cultured hepatocytes-abc transporter on canalicular membrane 8

9 Summary of BD Biosciences In Vitro Transporter Models Drug Transporter Model ABC Transporter SLC Transporter Non-cell based cell based cell based (OATP1B1 and OATP1B3 Vesicle Uptake assay Vesicle, Reagent Kit ATPase assay Membrane, Reagent Kit Hepatocytes Caco-2 Assay LLCPK- MDR1 Cells Transporter Qualified human CryoHepatocytes Suspension, Oil filtration kit Transporter Qualified Plateable Human CryoHepatocytes Fresh Human Platec Hepatocytes CryoHepatocytes: human, plateable, transporter qualified Fresh Hepatocytes: Human, plateable 9

10 ABC Transporter Assays ATPase Assay Vesicles Uptake Assay PGP ATP ADP + Pi 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 ATP Drug ADP + Pi BD Gentest ABC transporter vesicles Direct Assay: Measurement of transportermediated uptake of drugs into vesicles (substrates and inhibitors) Can be high throughput: using 96-well plate and cell harvester / vacuum manifold Assays developed for both radiolabeled and non-radiolabeled compounds 1

11 BD Gentest ABC Transporter Vesicles Inside Out Vesicles Human MRP1 Human MRP2 Human MRP3 Rat Mrp1 Rat Mrp2 Human BSEP Rat Bsep Human BCRP Rat Bcrp Mouse Bcrp Vesicles Assay Kit MRP/BCRP Vesicle Kit (cat. no. 4591) One kit supports all BD MRP and BCRP transporter vesicle assays 2 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 45911) Supports BSEP transporter vesicle assays 2 assays Probe substrate for BSEP 2 Year shelf-life Reagents only 1 X Wash Buffer for MRP/BCRP Vesicle Assay (cat. no 456) 1 X Wash Buffer for BSEP Vesicle Assay (cat. no 4561) 11

12 Vesicle Uptake Assays» Radiolabel Compound» Fluorescent Compound» Cold Compound 12

13 Overview of Assay Procedure Example of BSEP IC 5 Study Uptake activity with ATP Uptake activity with AMP ATP-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) IC 5 Calculation: 13

14 Vesicle Assay Procedure for using Radiolabeled Compound Scintillation Counter Reaction Pre-condition the filter plate Condition with: Stop reaction by transferring to vacuum manifold Wash the filter plate Vacuum Filter (facing down) 1) dh2o Dry the filter plate at room temp for 1-2 hrs Well Add 5 μl of scintillation liquid into each sample well LC-MS/MS analysis 14

15 Using Radio-labeled Compounds in Vesicle Uptake Assays Equipment Vacuum manifold/cell harvester, or alternative rapid filtration system 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 Quick solution for inhibition study Disadvantages Radiolabeled test articles are not always available for transporter phenotyping studies Limit the application spectrum Waste removal and radiation license requirements associated with radiolabeled compound Expensive 15

16 Vesicle Assay Procedure for using Fluorescent Compound Spectrophotometer Pre-condition the filter plate Condition with: 1) dh2o 2) 1X wash buffer 3) 1X blocking buffer Reaction Stop reaction by transferring to vacuum manifold Wash the filter plate Dry the filter plate at room temp for 1-2 hrs Elute into a new 96- well plate Elution Step: 1. Add 1 µl of.1n NaOH into each sample well 2. Incubate at Room temperature for 1 min 3. Elute by applying vacuum or centrifuge at 4 rpm for 5 min LC-MS/MS analysis 16

17 Using a Fluorescent Substrate in Vesicle Uptake Assays Equipment Vacuum manifold or alternative rapid filtration system spectrophotometer Compared with the physiologically relevant, high-affinity substrate, LTC 4, CDCF is: More robust MRP2 substrate, with similar kinetics as LTC 4 Optimal for high throughput screening of compound interactions No need for radiolabeled compound Requires a simple elution step in sample preparation Less expensive, more convenient, higher S/N ratio compared to using radiolabeled compound 17

18 Structure of 5(6)-Carboxy-2, 7 - Dichlorofluorescein (CDCF) Esterases CDA Fluorescent Ex: 485 nm Em: 538 nm 18

19 Time-Dependent Uptake of CDCF in MRP2/MRP3 Vesicle CDCF uptake in MRP/Mrp Vesicles (pmol/mg) human MRP2 S/N:4 S/N:3 S/N: rat Mrp2 S/N:9 S/N: human MRP3 S/N: Time (min) S/N:6 ATP-dependent uptake of CDCF is linear for at least 3 minutes for hmrp2/rmrp2, hmrp3 membrane vesicles. with ATP without ATP 19

20 Kinetic Study of CDCF in Human MRP Km of CDCF in MRP2 and MRP3 CDCF Uptake Activity (pmol/mg/min) human MRP2 rat Mrp2 human MRP CDCF Concentration (um) Vesicles K m values (μm) V max (pmol/mg/min) Average K m Average V max hmrp hmrp rmrp

21 CDCF is an Alternative to LTC 4 as an MRP2 Substrate Like LTC 4 (Leukotriene C 4 ), CDCF is transported by MRP2/Mrp2 in time-dependent, concentration dependent manner following Michaelis-Menten Kinetics. Comparison of IC 5 values of human MRP2 modulators. % of Remaining uptake Activity in hmrp LTC 4 as substrate Concentration of Inhibitor (μm) % of Remaining uptake Activity in hmrp CDCF as substrate Concentration of Inhibitor (μm) Indomethacin Benzbromarone MK571 Terfenadine Test compounds IC 5 (µm) Test compounds IC 5 (µm) Benzbromarone 7.8 Benzbromarone 3.3 MK MK Terfenadine 41.6 Terfenadine 45.8 Indomethacin Indomethacin

22 Cholyl-lysyl-fluorescein (CLF) CLF is a fluorescent bile salt derivative and has been developed as a potential agent for assessing in vivo liver function (Milkiewicz et al., 2). CLF uptake into human hepatocytes involves NTCP (Dawson et al, 29) and OATP1B1 and OATP1B3 (Hagenbuch, et al, 24). Indirect evidence demonstrates CLF is effluxed by BSEP, as same biliary excretion of CLF was observed in normal and TR(-) (Mrp2-deficient) Wistar rats (Mills, et al, 1999). CLF is going to be introduced as a novel agent for determination of in vivo liver function in a clinical setting. 22

23 Time-Dependent Uptake of CLF in Human MRP inside-out Vesicles 9 8 CLF Time-Dependent Uptake in MRP Inside-out Vesicles hmrp2 (S/N ~ 5) [CLF]:.8 μm Protein Conc. : 5 μg No GSH in reaction Control vesicle is used as control 7 CLF Uptake (pmol/mg) hmrp3 hmrp1 Control AMP Control ATP hmrp1 AMP hmrp1 ATP hmrp2 AMP hmrp2 ATP hmrp3 AMP hmrp3 ATP Incubation Time (min) 23

24 Time-Dependent Uptake of CLF in Human BSEP inside-out Vesicles 25 CLF Time-Dependent Uptake in BSEP Inside-out Vesicles hbsep (S/N ~ 2) [CLF]:.8 μm Protein Conc. : 5 μg Control vesicle is used as control 2 CLF Uptake (pmol/mg) 15 1 Control AMP Control ATP hbsep AMP hbsep ATP Incubation Time (min) 24

25 CLF Uptake in MRP2 is Saturable GSH has no effect on CLF uptake in human MRP2 vesicle CLF Uptake: Michaelis-Menten Kinetics K m = 2.7 μm 3 V max = pmol/min/mg AMP ATP Rate (pmol/min/mg) % of Inhibition of CLF Uptake Activity MK571 inhibition on CLF Uptake IC 5 = 29 μm MK571 Concentration (um) [Substrate] (µm). with GSH without GSH CLF uptake in human MRP2 vesicles is not stimulated by co-incubation with GSH. CLF uptake in human MRP2 vesicles follows Michaelis Menten Kinetics. CLF uptake in human BSEP vesicle is not saturable. 25

26 CLF Bile Accumulation is Inhibited by Cyclosporin A and MK571 5 μm CLF 5 μm CLF + 5 μm MK571 CLF Hepatic Uptake (Lot 295) CLF Uptake in Hepatocytes (pmol/mg) Incubation Time (min) [CLF]= 5μM 5 um CLF 5 um CLF + 1uM CsA MK571 is MRP2 inhibitor, can inhibit CDA bile excretion efficiently at concentration 5 μm. Cyclosporin A is inhibitor for OATP1B1/1B3, BSEP and MRP2 Hepatic uptake of CLF is mediated by OATP1B3, but not NTCP (Dirk R. de Waart, et al., JPET, 334:78-86, 21). 5 μm CLF 5 μm CLF + 1 μm Cyclosporin A 26

27 Summary Vesicle uptake assay using fluorescent substrate was validated. CDCF is a robust and convenient tool to characterize drug interactions with MRP transporters vs radiolabeled substrates. Uptake of CLF, a fluorescent bile salt derivative, in MRP2 vesicle is saturable and inhibited by MRP2 inhibitors. Our data indicated most likely MRP2 mediated biliary excretion of CLF. 27

28 Vesicle Assay Procedure for using Cold Compound LC-MS/MS analysis Pre-condition the filter plate Condition with: 1) dh2o 2) 1x wash buffer 3) 1x Blocking buffer Reaction Stop reaction by transferring to vacuum manifold Wash the filter plate Dry the filter plate at room temp for 1-2 hrs Elute into a new 96- well plate Elution Steps: 1.Add elution buffer with internal standard on filer plate 2.Incubate for 2 min at RT 3.Attach filter plate with a new 96- well receiver plate, centrifuge at 4 rpm for 5 min to collect the eluent 4.Analyze the sample on LC-MS/MS LC-MS/MS analysis 28

29 Using a Non-Radiolabeled (cold) Substrate in Vesicle Uptake Assays Equipment: Vacuum manifold or alternative rapid filtration system LC-MS/MS Advantage: Broad the application spectrum for all compounds Transporter substrate identification and inhibition study Can be used in any lab with analycal capability (LC-MS/MS) No radioactive hazard waste Disadvantage: Requires an elution step in sample preparation Compounds with strong non-specific binding on the filter device 29

30 Elution Buffer Optimization A LTC4 Uptake in hmrp2 Vesicle B E17bG Uptake in hmrp2 Vesicle Signal to Noise Ratio (S/N) Uptake Activity (pmol/mg/min) Signal to Noise Ratio (S/N) Uptake Activity (pmol/mg/min) 2 2 Elution 1 Elution 2 Elution 3 Elution 4 Elution 1 Elution 2 Elution 3 Elution 4 C S/N Uptake Activity E3S Uptake in hbcrp Vesicle S/N Uptake Activity Signal to Noise Ratio (S/N) Uptake Activity (pmol/mg/min) Elution Buffer: Elution 1: 8% Methanol; Elution 2: 8% Acetonitrile; Elution 3: 5 mm NaHCO3 in water; Elution 4: 5 mm NaHCO3 in 5/5 Methanol/Water. E lution 1 E lution 2 E lution 3 E lution 4 S/N U ptake A ctivity 3

31 Elution Time Optimization E17bG Transporter in MRP2 Vesicle Elution Time (min) 8% Methanol 5mM NaHCO3 in H2O LTC4 Transport in MRP2 Vesicle Elution Time (min) 8% Methanol 5mM NaHCO3 in H2O 31

32 Time-dependent Uptake of MRP2 Substrates LTC4 Uptake in hmrp2 E17bG Uptake in hmrp ATP AMP ATP AMP LTC4 Uptake (pmol/mg) E17bG Uptake (pmol/mg) Time (min) Time (min) 32

33 Time-dependent Uptake of BCRP Substrates MTX Uptake in hbcrp Chlorothiazide Uptake in hbcrp 1 3 MTX Uptake (pmol/mg) ATP AMP Chlorothiazide Uptake (pmol/mg) ATP AMP Time (min) Time (min) 33

34 Comparison of K m : LC-MS/MS vs Radiolabeled Compound LC-MS/MS Radiolabeled Uptake activity in MRP2 Vesicle (pmol/min/mg) K m : 148 M V max : 123 (pmol/min/mg) Hill coefficient : K m : 171 M V max : 145 (pmol/min/mg) Hill coefficient : 2.1 E17βG Uptake in hmrp2 Vesicle Incubation time: 5 min Protein Conc.: 5 M [Estradiol-17-Glucuronide] (µm) Uptake Activity in BCRP Vesicle(pmol/min/mg) K m : 7.2 M V max : 515 (pmol/min/mg) [E3S] (µm) ATP-dependent Uptake Activity of E3S (pmol/mg/min) [E3S] (μm) K m : 14.2±1.9 M V max : 56.±25.3 (pmol/min/mg) E3S Uptake in hbcrp Vesicle Incubation time: 3 min Protein Conc.: 5 M

35 Comparison of K m : LC-MS/MS vs Radiolabeled Compound 35

36 Vesicle Inhibition IC 5 Assay Radiolabeled vs Cold Substrates LC-MS/MS Analysis of E17bG Radiolabeled [3H] E 2 17BG MRP2 Substrate: E17βG Inhibitor: Benzbromarone % of Remaining E17bG Uptake Activity IC5=36 μm % of Remaining E17bG Uptake Activity IC5=36 μm Concentration of Benzbromarone (um) Concentration of Benzbromarone (µm) LC-MS/MS Analysis of E3S Radiolabeled [3H] E3S 1 12 BCRP Substrate: E3S Inhibitor: Sulfasalazine % of Remaining E3S Uptake Activity IC5=.4 μm % of Remaining E3S Uptake Activity IC5=.7 μm Concentration of Sulfasalazine (um) Concentration of Sulfasalazine (um) 36

37 Comparison of IC5: LC-MS/MS vs Radiolabeled Compound 37

38 Effect of Elution Buffer on Transport Kinetics and Inhibition Table 1 Effect of elution buffer on MRP2 substrate uptake kinetics Table 4 Effect of elution buffer on MRP2 inhibitor Benzbromarone IC 5 38

39 Summary The assay method was developed for inside-out vesicle uptake assay for non-radiolabeled chemicals using rapid filtration system and LC-MS/MS detection approach. Assay procedure was optimized for the selected MRP2 and BCRP substrates. There is no dramatic difference among the four elution buffers on the compound recovery, uptake ratio and kinetics in vesicle assay. Compound recovery is not affected by elution time significantly. The method was validated using kinetic assay of selected probe substrates and IC5 assay using selected inhibitors for both human MRP2 and BCRP transporters inside-out vesicles. The Km values and IC5 values determined by using LC-MS/MS were comparable with using radiolabeled compounds and literature report. 39

40 Summary of BD Biosciences In Vitro Transporter Models ABC Transporters Inside-out vesicles MRP/BCRP vesicle kit (4591); BSEP vesicle kit (45911) 1X wash buffer for MRP/BCRP vesicle assay (456) 1X wash buffer for BSEP vesicle assay (4561) Membranes ATPase assay kit (4596) Fresh Human Hepatocytes and Transporter Qualified Plateable Human CryoHepatocytes MDR1-LLPK cells/caco-2 cells SLC Transporters Hepatocyte suspension assay using oil-filtration method Uptake and inhibitory assays Fresh Human Hepatocytes and Transporter Qualified Plateable Human CryoHepatocytes 4

41 Q&A Questions? Contact information: Na Li, PhD Technical Support: tel: bdbiosciences.com/webinars For 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. 21 BD 41