PET/SPECT Imaging of Multidrug Resistance PET/SPECT Imaging of Multidrug Resistance P-glycoprotein:P-glycoprotein:

Transport Activity Monitoring Associated with Transport Activity Monitoring Associated with Blood-Brain Barrier FunctionBlood-Brain Barrier Function

David Piwnica-Worms, M.D., Ph.D.David Piwnica-Worms, M.D., Ph.D.

Director, Molecular Imaging CenterDirector, Molecular Imaging CenterMallinckrodt Institute of RadiologyMallinckrodt Institute of Radiology

and and Department of Developmental BiologyDepartment of Developmental BiologyWashington University Medical School Washington University Medical School

St. Louis, MOSt. Louis, MO

Molecular Imaging of Drug Transport In Vivo:Molecular Imaging of Drug Transport In Vivo:Multidrug Resistance (Multidrug Resistance (MDR1MDR1) P-glycoprotein) P-glycoprotein

Immunotech

-Efflux transporter-Efflux transporter-Normally expressed in liver, kidney, bowels,-Normally expressed in liver, kidney, bowels,

choroid plexus, capillaries of BBBchoroid plexus, capillaries of BBB-Pumps xenobiotics and cancer chemotherapeutic -Pumps xenobiotics and cancer chemotherapeutic

drugs out of MDR cancer cells and drugs out of MDR cancer cells and prevents drugs from entering brain via BBBprevents drugs from entering brain via BBB

-MDR drugs include: taxol, doxorubicin, -MDR drugs include: taxol, doxorubicin, vinblastine, vincristine, VP16, TKI’s vinblastine, vincristine, VP16, TKI’s (e.g., Gleevec), HIV protease inhibitors, (e.g., Gleevec), HIV protease inhibitors, antibiotics, antidepressants, peptides,antibiotics, antidepressants, peptides,lipophilic cationslipophilic cations

-Pumps -Pumps -amyloid out of the brain across BBB-amyloid out of the brain across BBB-MDR modulators (inhibitors) in clinical trials-MDR modulators (inhibitors) in clinical trials-MDR1 polymorphisms impact drug PK/PD -MDR1 polymorphisms impact drug PK/PD

Probable Model for TransportProbable Model for Transportof Recognized Substratesof Recognized Substrates

Science 2009, 323, 1718-1723Science 2009, 323, 1718-1723

Front ViewFront View Back ViewBack View

Crystal Structure: Mouse P-GlycoproteinCrystal Structure: Mouse P-Glycoprotein

Radiopharmaceuticals Targeting Radiopharmaceuticals Targeting MDR1MDR1 P-glycoprotein P-glycoprotein

99m99mTc-SestamibiTc-Sestamibi99m99mTc -TetrofosminTc -Tetrofosmin99m99mTc-Q58, Tc-Q58, 99m99mTc-Q63Tc-Q6399m99mTc-CO-mibiTc-CO-mibi6767Ga-ENBPI Ga-ENBPI

6868Ga-ENBPI Ga-ENBPI 6464Cu-dioximeCu-dioxime1111C-verapamilC-verapamil1111C-daunorubicinC-daunorubicin1111C-colchicineC-colchicine94m94mTc-SestamibiTc-Sestamibi1818F-paclitaxolF-paclitaxol1111C-loperamideC-loperamide

SPECT AgentsSPECT Agents PET AgentsPET Agents

C

C

C

C

C

C

N

NN N

NN

O M e

M e O

O M e

M e O

M e O

M e O

P

P

P

P

O E t

O E tO E t

E t O

O E tO E t

O E tE t O

O

O

N N

P

P

T c

O M eM e O

O M e

O M eM e O

M e O

OOO O

N N

P

P

T c

OO

O M eM e O

O M eM e O

M e O O M e

O M eM e O

Tc

Tc-99m-Sestamibi Tc-99m-Tetrofosmin Tc-99m-Furifosmin Tc-99m-Q58

Tc

Tc-99m-Based Agents

O

O

1 1C H3

O

O

O

N H2O H

O M e

O H

O H

O HN H C O C H3

O1 1C H3

O

M e O

M e O

O M e

N

N O

O N H

N H

O M eM e O

O M eM e O

G a

C-11-Colchicine C-11-Daunorubicin Ga-68-4,6-DiMeO-ENBPI

Positron Emission Tomography Agents

Conventional Radiopharmaceuticals Targeting Conventional Radiopharmaceuticals Targeting MDR1MDR1 P-glycoprotein P-glycoprotein

Conventional SPECT Agents Recognized by Conventional SPECT Agents Recognized by MDR1MDR1 Pgp Pgp as Transport Substratesas Transport Substrates

C

C

C

C

C

C

N

NN N

NN

OMeMeO

OMe

MeO

MeO

MeO

P

P

P

P

OEt

OEtOEt

EtO

OEtOEt

OEtEtO

O

O

N N

P

P

Tc

OMeMeO

OMe

OMeMeO

MeO

OOO O

N N

P

P

TcOO

OMeMeO

OMeMeO

MeO OMe

OMeMeO

Tc-99m-Sestamibi

TcTc

Tc-99m-Tetrofosmin

Tc-99m-Furifosmin Tc-99m-Q58

New SPECT Agent Recognized by New SPECT Agent Recognized by MDR1MDR1 Pgp Pgp as a Transport Substrateas a Transport Substrate

67GaN

HN NH

N

O

O

O

O

67Ga-ENBPI

Robert Innis, et al.Robert Innis, et al.

New PET Agent Recognized by New PET Agent Recognized by MDR1MDR1 Pgp Pgp as a Transport Substrateas a Transport Substrate

1111C-LoperamideC-Loperamide

MODEL OF [MODEL OF [99m99mTc]-SESTAMIBI ACCUMULATION Tc]-SESTAMIBI ACCUMULATION IN TUMOR CELLS:IN TUMOR CELLS:

•MEMBRANE POTENTIAL-DRIVEN PASSIVE INFLUXMEMBRANE POTENTIAL-DRIVEN PASSIVE INFLUX• MDR1MDR1 P-GLYCOPROTEIN-MEDIATED EFFLUX TRANSPORT P-GLYCOPROTEIN-MEDIATED EFFLUX TRANSPORT

Tc-ComplexTc-Complex

Tc-ComplexTc-Complex

mitomito

cell membranecell membrane

Cell UptakeCell Uptake

maxmax

minmin

wtwt

ADPADP

ATPATP

MDR1MDR1 P-glycoprotein P-glycoproteinTc-ComplexTc-Complex MDRMDR

High Potency MDR High Potency MDR InhibitorsInhibitors::

Glaxo GG918Glaxo GG918Novartis PSC 833Novartis PSC 833

Vertex 710Vertex 710Eli Lilly 335979Eli Lilly 335979

XR9576XR9576

((--))

MDRMDR+ Inh+ Inh

Mitochondrial rich tissue; No P-glycoprotein expressionMitochondrial rich tissue; No P-glycoprotein expression

Electron Density Map Technetium MapElectron Density Map Technetium Map

Electron Probe X-ray Microanalysis (EPXMA)Electron Probe X-ray Microanalysis (EPXMA) of a Cultured Heart Cell Incubated in Technetium-99-Sestamibiof a Cultured Heart Cell Incubated in Technetium-99-Sestamibi

6060505040403030202010100000

100100

200200

300300

TIME (min)TIME (min)

Tc-S

ESTA

MIB

I CO

NTE

NT

( fm

ol /

mg

prot

ein

nM

)Tc

-SES

TAM

IBI C

ON

TEN

T ( f

mol

/ m

g pr

otei

n n

M ) oo

WILD TYPE VIRUSWILD TYPE VIRUS

RECOMBINANT RECOMBINANT MDR1MDR1

Sf9 MDR1 WTSf9 MDR1 WT

Pgp-Pgp-

Baculoviral Expression of Recombinant Human Baculoviral Expression of Recombinant Human MDR1MDR1 in Host Sf9 in Host Sf9 Cells:Cells:

Effect on [Effect on [99m99mTc]-Sestamibi AccumulationTc]-Sestamibi Accumulation

[[99m99mTc]-SestamibiTc]-Sestamibi

Pgp

ABC Transporter-Mediated Efflux of [ABC Transporter-Mediated Efflux of [99m99mTc]-SestamibiTc]-SestamibiIn Cultured Cell Monolayer AssaysIn Cultured Cell Monolayer Assays

High Avidity TransportHigh Avidity TransportMDR1 PgpMDR1 Pgp

Modest Avidity TransportModest Avidity TransportMRP1MRP1

Minimal or Non-detectable TransportMinimal or Non-detectable TransportMDR3 PgpMDR3 PgpMRP2, MRP3, MRP4, MRP5, MRP6MRP2, MRP3, MRP4, MRP5, MRP6BCRP1/MXRBCRP1/MXR

Imaging MDR1 P-glycoprotein Transport Activity in Breast Cancer: Dynamic [99mTc]-Sestamibi Mammoscintigraphy

11 Tumor Tumor

1 hr post-injection1 hr post-injection

4 hr post-injection4 hr post-injection

[[99m99mTc]-SestamibiTc]-Sestamibi

Del Vecchio, et al, Eur J Nucl Med 24:150, 1997

[[99m99mTc]-Sestamibi Clearance From Breast Cancers Tc]-Sestamibi Clearance From Breast Cancers In VivoIn Vivo

Low Pgp

High Pgp

Pgp Expressed Only on the Luminal Surface of Brain Capillaries:Pgp Expressed Only on the Luminal Surface of Brain Capillaries:Provides Unidirectional Drug Transport Function at the BBBProvides Unidirectional Drug Transport Function at the BBB

CSFCSFBloodBlood

Capillary Endothelial CellsCapillary Endothelial Cells

PgpPgp

BBBBBB

Compound EffluxCompound Efflux

Drug BarrierDrug Barrier

T1 MRI Tc-MIBI SPECTCo-Registration

Functional Demonstration of the Drug-Permeability Barrier at the BBB in Humans with 99mTc-Sestamibi

PNAS, 1999PNAS, 1999

Functional Demonstration of the Drug-Permeability Barrier Functional Demonstration of the Drug-Permeability Barrier at the BBB in Humans with at the BBB in Humans with 1111C-VerapamilC-Verapamil

Sasongko, et al, 2005Sasongko, et al, 2005

Robert Innis, et al., 2009Robert Innis, et al., 2009

Lop

1111C-C-N-DesmethylN-Desmethyl-Loperamide-Loperamide

Functional Demonstration of the Drug-Permeability Barrier Functional Demonstration of the Drug-Permeability Barrier at the BBB in Humans with at the BBB in Humans with 1111C-Loperamide C-Loperamide

0 20 40 60 80 100 120 %

Inje

cted

Dos

e[T

issu

e C

i(Inj

ecte

d C

i)-1g-1

x 1

00]

0.0

0.5

1.0

1.5

Time (min)0 20 40 60 80 100 120

0.0

0.2

0.4

0.6

0.8

0 20 40 60 80 100 1200

10

20

30Blood Brain Liver

MicroPET: 10 min p.i.MicroPET: 10 min p.i.

brain

WT mdr1a/1b -/-

Biodistribution AnalysisBiodistribution Analysis

mdr1a/1b -/-

WT

Molecular Imaging of Molecular Imaging of mdr1mdr1 P-glycoprotein Transport Activity P-glycoprotein Transport Activity at the Capillary Blood-Brain Barrier at the Capillary Blood-Brain Barrier in vivoin vivo with ENBPI Ga-Complexes: with ENBPI Ga-Complexes:

Correlation between Biodistribution and MicroPET AnalysisCorrelation between Biodistribution and MicroPET Analysis

6868Ga-3EtO-ENBPIGa-3EtO-ENBPI

Bidirectional Dynamic Model for Influx and Efflux Bidirectional Dynamic Model for Influx and Efflux of of -Amyloid (A-Amyloid (Aβ) β) Across the BBB:Across the BBB:

Pgp Transports Pgp Transports -Amyloid out of the CNS-Amyloid out of the CNS

mdr1mdr1 P-glycoprotein Deficiency at the Blood Brain Barrier Increases P-glycoprotein Deficiency at the Blood Brain Barrier Increases -Amyloid-Amyloid Deposition in an Alzheimer Disease Mouse Model: Deposition in an Alzheimer Disease Mouse Model:

P-glycoprotein Pumps P-glycoprotein Pumps -Amyloid-Amyloid out of the Brain out of the Brain

J Clin Invest 115: 3285, 2005J Clin Invest 115: 3285, 2005Enhanced amyloid plaque content in aged APPsw, Pgp-null miceEnhanced amyloid plaque content in aged APPsw, Pgp-null mice

Pgp-null mice microinjected into CNS with Pgp-null mice microinjected into CNS with -Amyloid-Amyloid::Enhanced retention and reduced clearance from the brain Enhanced retention and reduced clearance from the brain

Acute inhibition of Pgp with XR9576:Acute inhibition of Pgp with XR9576:Enhancement of CNS Enhancement of CNS -Amyloid-Amyloid content content

NanoSPECT Imaging: Comparative Uptake of NanoSPECT Imaging: Comparative Uptake of 99m99mTc-Sestamibi inTc-Sestamibi in APPsw/PS1-PgpWT and APPsw/PS1-Pgp-Null Mice APPsw/PS1-PgpWT and APPsw/PS1-Pgp-Null Mice

2h Post Injection; 3-months old mice

APPsw/PS1-PgpWTAPPsw/PS1-PgpWT APPsw/PS1-Pgp NullAPPsw/PS1-Pgp Null

Brain

Molecular Imaging of Multidrug Resistance (Molecular Imaging of Multidrug Resistance (MDR1MDR1) P-glycoprotein (Pgp) ) P-glycoprotein (Pgp) Transport Activity at the BBBTransport Activity at the BBB

LOW Pgp

HIGH Pgp

Tc-99m-Sestamibi

PgpPgp

Pgp pumps drugs and substratesPgp pumps drugs and substratesoff the plasma membraneoff the plasma membrane

[[99m99mTc]-SestamibiTc]-Sestamibi

Several PET and SPECT tracers show promiseSeveral PET and SPECT tracers show promise as transport substrates for monitoring Pgpas transport substrates for monitoring Pgp

Pgp is an important component of the BBB Pgp is an important component of the BBB and can be functionally imaged by and can be functionally imaged by PET and SPECTPET and SPECT

Pgp pumps Pgp pumps -Amyloid out of the brain across-Amyloid out of the brain acrossthe BBB. Pgp polymorphisms may bethe BBB. Pgp polymorphisms may bean imagable risk factor for AD.an imagable risk factor for AD.

Seth GammonSeth Gammon

Piwnica-Worms Lab, Molecular Imaging CenterPiwnica-Worms Lab, Molecular Imaging Center

Funding: NIH P50, RO1, DODFunding: NIH P50, RO1, DOD

VijayVijaySharmaSharma