Imaging Techniques and Pharmacokinetic … L. R. Brouwer School of Pharmacy, University of North...

Time (min)0 20 40 60 80 100 120 140 160 180

Tc-9

9m m

ebro

feni

n (%

dos

e)0.1

1

10

100

Tc-9

9m m

ebro

feni

n (%

dos

e)

0

20

40

60

80

100Blood Bile Liver Urine

Kim L. R. Brouwer

School of Pharmacy,

University of North Carolina at Chapel Hill

Imaging Techniques and Pharmacokinetic Modeling/Simulation:

Useful Quantitative Tools for the Drug Transport Scientist

X0 Xbile

Xurine

k10

Cc

XliverVc

k12

k21

k20X0 Xbile

Xurine

k10

Cc

XliverVc

k12

k21

k20

Kim L. R. Brouwer

Eshelman

School of Pharmacy,

University of North Carolina at Chapel Hill

Imaging Techniques and Pharmacokinetic Modeling/Simulation

Available Tools–

Utility

–

Requirements–

Advantages

–

LimitationsWhen should these tools be used? What questions can be addressed?How can in vivo, in situ and in vitro drug transport data, coupled with modeling and simulation, be used to predict the impact of altered transport on drug disposition?

Whole Body Autoradiography (WBA)Utility: Provides general view of distribution pattern of compound in tissues/organs, preliminary information on ADME properties of radiolabeled compound, correlation with observed toxicities, presence of biliary excretion or direct intestinal secretionRequirements: Radionuclides (3H or 14C; 50-100 μCi) administered i.v. or p.o. to preclinical species; animals frozen and mounted; 20 micron sections freeze dried, exposed to phosphor imaging plates, and quantified with imaging analysis or directly by LCMSAdvantages: Provides information regarding organ distribution, unforeseen localization of radioactivity and new routes of eliminationLimitations: Monitors distribution of total radioactivity and can’t discriminate between parent and metabolites

Use of WBA to Assess Pgp Inhibition of 3H-Digoxin Distribution

in Wild-Type and MDR 1a/1b -/- MiceWild-Type Mice Mdr1a/1b -/-

Mice

90min

4 hour

8 hour

24 hour

Courtesy of Glaxo, Inc.

AntiviralAlone

Antiviral +GF120918 CSF

Brain

CSFBrain

Blood

Ratio

Antiviral Alone

Antiviral + GF120918

Brain/Blood

CSF/Blood

0.35

0.86

0.06

0.45

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

Ratio

Antiviral Alone

Antiviral + GF120918

Brain/Blood

CSF/Blood

0.35

0.86

0.06

0.45

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

Ratio

Brain/BloodCSF/Blood

Use of WBA to Assess Effect of GF120918 on Antiviral Distribution in Rats

Courtesy of Glaxo, Inc.

Positron Emission Tomography (PET)

Utility: Noninvasive imaging method to quantify activity of transport proteins in preclinical species or humans, or determine the functional impact of transport protein modulation associated with genetic polymorphisms, transport inhibition or inductionRequirements: PET probes for specific transport proteins; typical isotopes: 11C (~20 min); 13N (~10 min); 18F (~110 min)Advantages: Accurately measure amount of tracer accumulation in organ over time; direct measurement of transport protein activity in species of interest; noninvasiveLimitations: Availability of specific, labeled probes; synthesis of radiolabeled compounds- due to short half-lives, cyclotron and radiochemistry lab must be in close proximity to PET imaging facility

Use of PET to Evaluate Pgp Function at the BBB in Humans:

Inhibition of [11C]Verapamil Transport by Cyclosporine

Sasongko

et al., Clin Pharmacol Ther 77:503, 2005

Celecoxib Does Not Affect Pgp-Mediated Efflux of [11C]Verapamil in Mice

de Vries

et al., Nucl Med Biol 35:459, 2008

Untreated Celecoxib CsA

Gamma ScintigraphyUtility: Noninvasive imaging method to quantify activity of transport proteins in preclinical species or humans, or determine the functional impact of transport protein modulation associated with genetic polymorphisms, transport inhibition or inductionRequirements: Short-lived gamma emitting radioisotopes that are probes for specific transport proteins [e.g., 99mTc-Mebrofenin, 99mTc-Sestamibi)]Advantages: Accurately measure amount of tracer accumulation in organ over time; direct measurement of transport protein activity in species of interest; noninvasive; t1/2 more reasonable than PET probesLimitations: Availability of specific, labeled probes; quantitation issues

Relationship Between SN-38 AUC and99mTc-Sestamibi (MIBI) Hepatic Retention

Michael

et al. J. Clin. Oncol. 24:4228, 2006

Relationship Between Hepatic Nuclear Imaging Parameters and Irinotecan

Pharmacokinetics and ToxicityA significant, linear correlation was observed between SN-

38 AUC and the percent retention at 1 hr of 99mTc- iminodiacetic

acid and 99mTc-

sestamibi. Data suggest that:

–

A higher grade of neutropenia

was associated with lower biliary excretion

Neutropenia has been positively correlated with SN-38 plasma concentrationsReduced hepatic tracer clearance may be associated with increased SN-38 exposure

–

Increased diarrhea was reported in patients with higher biliary transport function

Late onset diarrhea has been attributed to SN-38 intestinal secretion

Michael

et al. J. Clin. Oncol. 2006 24:4228

Application of Nuclear Imaging to Evaluate Biliary Clearance in Humans

Sphincter of Oddi

Pancreas

Ligament of Treiz

Common bile duct

Right hepatic duct Left hepatic

duct

Cystic duct

Stomach

Duodenum

Intestinal Hepatic Renal

Biliary Metabolic

Drug Clearance

•

Clbiliary

may contribute significantly to Clhepatic•

Altered Clbiliary due to genetics, disease states or drug interactions may affect pharmacological efficacy and/or toxicity (systemic, hepatic and/or intestinal)

•

Species differences in hepatic transport proteins may limit the utility of animal models to predict human Clbiliary

Oro-enteric Tube and Protocol Design

Multi-lumen silicone extrusion Double duodenal aspiration channelsGastric aspiration channelOcclusive balloon (positioned in the distal duodenum by fluoroscopy)

Fatty meals to maintain gallbladder contractilityIV administration of 99mTc radiotracersCCK8 IV infusion to contract gallbladderGamma scintigraphy of abdominal area

Ghibellini

G et al. AAPS Journal, 6 (4)

Article 33, 2004

A C B

122 cm115 cm110 cm

AB

C

D

E

F

0 cm 85 cmgastric aspiration holes

95 cm

radio-opaque marker

Saline flush outlet105 cm

duodenal aspiration channel 1

128 cm

Tungsten tip-weightsPolyethylene balloon inflation/deflation ports

Polyethylene balloon attachment points

duodenal aspiration channel 2

100 cm4.5 mm

A = radio-opaque marker at pyloric sphincterB = balloon attachment ringsC = tungsten tip weights at Ligament of Treitz

Calculation of Parameters and Corrected Biliary

Clearance

XGB Excreted Dose = corrected amount of drug collected from the duodenum when accounting for incomplete gallbladder (GB) contraction

Based on counts from frontal planar image of GBNormal EF > 35%

Tlast0

GBbiliary

Tlast0

Tlastbile0biliary

0total

AUCXCl vivo in

AUCXCl

AUCDoseCl

−

−

−

∞−

=

=

=

( )preCCK

postCCKpreCCK

minymin120bilemin120bileGB

GBGBGB

EF

EFXXX

−=

⎟⎠⎞

⎜⎝⎛+= −

−

Time (min)

GB

RO

I cou

nts

per s

econ

d

Time (min)

GB

RO

I cou

nts

per s

econ

d

Ejection Fraction (EF) Curves

y time over which the GB ejection fraction (EF)was calculated

99mTc-MebrofeninClinical Use: hepatobiliary imaging agentLiver uptake: 98%

HNN

O

OO

99mTc3+

O O

N

O

O

OO Br

NH

OBr

-

N

MeO

C

N

OMe

C N

OMe

C

N OMeC

OMe

CN

MeO

C

N

99mTc

+99mTc- SestamibiClinical Use: myocardial perfusion agentSubstrate for P-gp, MRP1, BCRP and Mrp2 30-40% of dose excreted in feces; 27% in urine

Probe Compounds

Gamma Scintigraphic Images (0-180 min) of 99mTc-Mebrofenin Disposition

Spontaneous and prolonged GB emptying into duodenum with successful aspiration of secretions

Strong GB response to CCK8 and complete collection of bile

Balloon positioned very close to Sphincter of Oddi

38 min 75 min

Ghibellini

et al. AAPS Journal (2004); 6 (4)

Article 33

99mTc-Mebrofenin Disposition *Recovery expressed as % of dose Cl values expressed as mL/min/kg

Parameter Mean S.D.

Dose (µCi) 2277 292

*Biliary Recovery 67.1 23.0

*Urinary Recovery 0.6 0.2

Ejection Fraction 0.64 0.4

*Recovery of Excreted Dose 84.2 9.3

Cltotal 17.3 1.7

Clbiliary 12.5 3.6

in vivo Clbiliary 16.1 3.2

Spontaneous and CCK8 stimulated GB emptying

Intestinal occlusion and complete collection of bile were achieved

In vivo Clbiliary similar to human liver blood flow (21 ml/min/kg)

Ghibellini

et al. AAPS Journal (2004); 6 (4)

Article 33

X0 Xbile

Xurine

k10

Cc

XliverVc

k12

k21

k20X0 Xbile

Xurine

k10

Cc

XliverVc

k12

k21

k20

All processes are linear and 1st -orderRenal clearance solely via glomerular filtrationModel selection based on: AIC, RSS, visual inspection of residual plots and of model fit to data

Parameter Mean S.D.

K12 (min-1) 0.18 0.06

K21 (min-1) 0.0034 0.0013

K20 (min-1) 0.012 0.003

K10 (min-1) 0.0009 0.0004

Vc (L) 9.56 3.25

Ratio K20/K21 3.5 0.8 Time (min)

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9m m

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1

10

100

Tc-9

9m m

ebro

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n (%

dos

e)

0

20

40

60

80

100Blood Bile Liver Urine

Pharmacokinetic Model to Describe99mTc-Mebrofenin Disposition in Humans

Ghibellini

et al. Pharm Res, 25:1851, 2008

Liver Disease

Liver Disease Simulations:

–

Mild Hyperbilirubinemia:k12

↓

2.5-fold

–

Intermediate Hyperbilirubinemia:k12

↓

5-fold

–

Inflammation-induced Cholestasis:k12

↓

5-fold and k20

↓

2-fold

–

Obstructive Cholestasis:k21

↑5-fold and k20

↓

3-fold

Blood

X0 Xbile

Xurine

k10

Cc

XliverVc

k12

k21

k20X0 Xbile

Xurine

k10

Cc

XliverVc

k12

k21

k20

Relevance• Major differences in hepatic exposure are

evident when biliary excretion is impaired

Liver

Normal

IntermediateHyperbilirubinemiaMildHyperbilirubinemia

Obstructive Cholestasis

Inflammation-InducedCholestasis

Ghibellini

et al. Pharm Res, 25:1851, 2008

Gamma Scintigraphic Images (0-180 min) of 99mTc-Sestamibi Disposition

5 min 12 min 29 min 99 min

143 min125 min 180 min169 min

A

AAA

AA

AB B

RKLK

SP

HT

BL

LV

5 min 12 min 29 min 99 min

143 min125 min 180 min169 min

5 min 12 min 29 min 99 min5 min5 min 12 min12 min 29 min29 min 99 min99 min

143 min125 min 180 min169 min143 min143 min125 min125 min 180 min180 min169 min169 min

A

AAA

AA

AB B

RKLK

SP

HT

BL

LV

Ghibellini

et al., Clin Pharmacol Ther, 81:406, 2007

Dotted oval outline: balloonArrow A: intestinal secretion of 99mTc-sestamibi No visible contraction of GB nor radioactivity recovered from duodenal aspirates until CCK8 administration (120 min)No additional radioactivity in the GI tract below the balloon after CCK8 indicating successful occlusion of the intestine

Sandwich-Cultured HepatocytesUtility: Evaluation of mechanisms and extent of hepatic uptake, basolateral excretion and biliary excretion of drugs and derived metabolites in species of interest; evaluation of localization, trafficking and regulation of basolateral and canalicular transport proteinsRequirements: Freshly isolated hepatocytes or transporter-competent cryopreserved hepatocytesAdvantages: Hepatocytes cultured between 2 layers of gelled collagen establish intact canalicular networks, express relevant hepatic transport proteins, re-establish polarized excretion of compounds, and represent a useful model to investigate hepatobiliary dispositionLimitations: Requires 3-4 days for proper localization of canalicular transport proteins

Pre-isolation 0 hours 24 hours 48 –

96 hours

Percoll Gradient85 – 95% Viability

Liver Perfusion (~35 ml/min, 37oC):10 min Ca2+-free with chelator10 min collagenase digestionSingle pass or recirculating flow

Hepatocyte isolationLiver capsule gently torn

Sandwich-Cultured Hepatocytes: Experimental Procedures

Liu et al., Am J Physiol 227:G12, 1999

Zamek-Gliszczynski and Brouwer. In Pharmaceutical Profiling in Drug Discovery for Lead Selection. AAPS Press, 2004

Day 1 Day 6Day 1 Day 6

MRP2

1 3 4 6Days in Culture

Expression of MRP2 in SC human hepatocytes over days in culture

Immunohistochemical co-localization of MRP2 (green) andMDR1 (red) in Day 6 SC human hepatocytes

Fluorescence of 5 (and 6)-carboxy-2,7-dichlorofluorescein in Day 1 and Day 6 SC human hepatocytes after incubation with 2 µM CDF diacetate for 10 min

MRP2 Expression, Localization and Function in Sandwich-Cultured (SC) Human Hepatocytes

Hoffmaster et al., Pharm Res 21:1294, 2004

Quantitation of Biliary Excretion in Sandwich-Cultured Hepatocytes (B-CLEAR®)

Substrate inBile Canaliculi

(bc)

Standard Buffer

cells bc cells

Ca2+ -free Buffer

cells cells

B-CLEAR®

is covered by US Pat. No. 6,780,580 and other US and International patents both issued and pending.

Biliary Excretion Index (%)

In Vitro Biliary Clearance

Accumulation cells + bc -

Accumulation cells

Accumulation

cells + bc= x 100

Accumulation cells + bc -

Accumulation cells

Timeincubation x Concentration

medium=

Sandwich-Cultured Human Hepatocytes

Human hepatocytes from 4 living donors cultured in sandwich configuration for 6 days

On Day 6: taurocholate accumulation measured as marker of cell functionality

If taurocholate BEI ≥ 50% then further experiments were conducted

0

50

100

150

200

250

Liver 2 Liver 3 Liver 4Liver 1

Acc

umul

atio

n (p

mol

/mg

prot

ein)

BEI=50% BEI=55%

BEI=56%

BEI=66%

0

50

100

150

200

250


Acc

umul

atio

n (p

mol

/mg

prot

ein)

BEI=50% BEI=55%

BEI=56%

BEI=66%


Acc

umul

atio

n (p

mol

/mg

prot

ein)

BEI=50% BEI=55%

BEI=56%

BEI=66%

cells+bilecells

Time (min)0 2 4 6 8 10

[3 H]T

C U

ptak

e(p

mol

/mg

prot

ein)

0

100

200

300

400

500cells + bccells

Taurocholate Accumulation in Sandwich-Cultured Human Hepatocytes

3H-Taurocholate

Ghibellini

et al., Clin Pharmacol Ther,81:406, 2007

Day 6 Sandwich-Cultured Human Hepatoctyes

0

5

10

15

20

25

30

BEI=34%

BEI=20%

BEI=31%

BEI=54%


Acc

umul

atio

n (%

dos

e/ m

g pr

otei

n)

0

5

10

15

20

25

30

BEI=34%

BEI=20%

BEI=31%

BEI=54%


Acc

umul

atio

n (%

dos

e/ m

g pr

otei

n)

BEI=34%

BEI=20%

BEI=31%

BEI=54%


Acc

umul

atio

n (%

dos

e/ m

g pr

otei

n)99mTc-Mebrofenin

0.0

0.1

0.2

0.3

0.4


Acc

umul

atio

n (%

dos

e/ m

g pr

otei

n)

BEI=13%

BEI=9%

BEI=29% BEI=27%

0.0

0.1

0.2

0.3

0.4


Acc

umul

atio

n (%

dos

e/ m

g pr

otei

n)

BEI=13%

BEI=9%

BEI=29% BEI=27%

Piperacillin0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

BEI=32%

BEI=23%

BEI=40%

BEI=38%


Acc

umul

atio

n (%

dos

e/ m

g pr

otei

n)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

BEI=32%

BEI=23%

BEI=40%

BEI=38%


Acc

umul

atio

n (%

dos

e/ m

g pr

otei

n)

99mTc-Sestamibi

99mTc-mebrofenin accumulation over 10 min (0.5 μCi/mL)

99mTc-sestamibi accumulation over 10 min (0.5-5 μCi/mL)

Piperacillin accumulation over 30 min (300 μM)

cells+bilecells

Ghibellini

et al., Clin Pharmacol Ther, 81:406, 2007

In Vitro-In Vivo Correlation of Clbiliary Corrected for Hepatocellularity Per Gram Liver in Humans

Ghibellini

et al., Clin Pharmacol Ther, 81:406, 2007Barter et al., Curr Drug Metab, 8:33, 2007

0.003

0.03

-0.3

3.0

30

0.003 0.03 - 0.3 3.0 30

Pred

icte

d B

iliar

y C

L(m

l/min

/kg)

Observed Biliary CL(ml/min/kg)

Tc-99m Mebrofenin

Tc-99mSestamibi

Piperacillin

Imaging Techniques and Pharmacokinetic Modeling/Simulation

Available Tools–

Utility

–

Requirements–

Advantages

–

LimitationsWhen should these tools be used? What questions can be addressed?How can in vivo, in situ and in vitro drug transport data, coupled with modeling and simulation, be used to predict the impact of altered transport on drug disposition?

Use of Pharmacokinetic Modeling to Investigate the Hepatobiliary Disposition of APAP, AG and AS

1

10

100

0 30 60 90 120 150

0.1

1

10

100

0 30 60 90 1200.1

1

10

100

0 30 60 90 120

1

10

100

0 30 60 90 120 150

A B

C D

Control TR-

Time (min)

Con

cent

r atio

n ( μ

g/m

l)

Con

cent

ratio

n (μ

g/m

l)

Time (min)

Excr

etio

n R

ate

(μg/

min

)

Time (min) Time (min)

Exc

retio

n R

ate

(μg/

min

)

Xiong, H. et al., J Pharmacol Exp Ther 295:512-518, 2000

APAP

(V) KAG KmAS VmaxAS KPAG KBAG

AG AG AG (VR) KPAS KBAS AS AS AS CLAS Perfusate Hepatocyte Bile

x

Pharmacokinetic Model for Isolated PerfusedTR- (Mrp2-deficient) Rat Livers


a Mean ±

SD, n = 4 for control group, n = 5 for TR-

group b KmAS was assumed to be 17 μg/ml (Tone et al., 1990)* P < 0.05

Pharmacokinetic Parameters Describing the Hepatobiliary Disposition of APAP, AG and AS

CONTROL TR-

KOTHER (min-1) 0.003 ± 0.001 N/A

KAG (min-1) 0.007 ± 0.001 0.010 ± 0.003

VmaxAS (μg/ml/min/g liver) 0.106 ± 0.037 0.143 ± 0.036

KPAG (min-1) 0.028 ± 0.004 0.206 ± 0.095*

KBAG (min-1) 0.027 ± 0.016 N/A

KPAS (min-1) 0.115 ± 0.051 0.108 ± 0.058

KBAS (min-1) 0.007 ± 0.003 0.0005 ± 0.0002*

CLAS (ml/min/g liver) 0.045 ± 0.013 0.132 ± 0.073

V (ml) 92.8 ± 5.7 95.3 ± 8.7

~7.4 fold


0 30 60 90 1200

10

20

30

40

AG

Per

fusa

te C

onc.

(μg/

ml)

Time (min)

— KPAG , 0.206 min-1

— KPAG , 0.028 min-1

Increased Basolateral Egress of AG in TR- Rat Livers:Simulated AG Perfusate Concentration vs. Time Profile

APAP

(V) KAG KmAS VmaxAS KPAG KBAG

AG AG AG (VR) KPAS KBAS AS AS AS CLAS Perfusate Hepatocyte Bile


Schematic Diagram of the PBPK Model Predicting Pravastatin Disposition

Watanabe

et al., J Pharmacol Exp Ther, 328:652-662, 2009

Effects of Changes in Transporter Activity on Plasma and Liver (Target Organ)

Concentrations of Pravastatin in Humans

Watanabe

et al., J Pharmacol Exp Ther, 328:652-662, 2009

Plasma

Liver

PSinf PSbile

- - - x 1/3....... x 3

- - - x 1/3....... x 3

- - - x 1/3....... x 3

- - - x 1/3....... x 3

Time (min)0 20 40 60 80 100 120

Acc

umul

atio

n in

Med

ia (p

mol

)

10

100

1000

10000

Disposition of Troglitazone and Metabolites in Sandwich-Cultured Rat Hepatocytes

T im e (m in )0 20 40 60 80 100 120

Acc

umul

atio

n in

Cel

l (pm

ol)

1

10

100

1000

10000

TGZ

TS

TG

TQ

TS

TGZ

TG

Medium Hepatocyte

Lee

et al., AAPS Transport Workshop, 2009

Impact of Kbile,TS Modulation on Troglitazone Sulfate (TS) Accumulation in Sandwich-Cultured Rat Hepatocytes

0 20 40 60 80 100 120

010

0020

0030

0040

00

0 20 40 60 80 100 120

010

0020

0030

0040

00

0 20 40 60 80 100 120

010

0020

0030

0040

00

0 20 40 60 80 100 120

010

0020

0030

0040

00

0 20 40 60 80 100 120

010

0020

0030

0040

00

Control10X higher2X higher 10X lower2X lower

Time (min)

Acc

umul

atio

n of

TS

in C

ell (

pmol

)

Lee

et al., AAPS Transport Workshop, 2009

Conclusions•

Numerous imaging methods (WBA, PET, Gamma Scintigraphy) may be used to examine drug distribution and quantify activity of transport proteins

•

Nuclear imaging agents (99mTc-Mebrofenin; 99mTc-Sestamibi), coupled with use of a specialized oroenteric tube and clinical protocol, can be used to quantify Clbiliary

of drugs in vivo in humans

•

Application of gamma scintigraphy

in drug development may provide insight regarding:–

drug-drug interactions in hepatobiliary transport–

intestinal secretion of drugs and/or metabolites

In vivo, in situ and in vitro drug transport data, coupled with modeling and simulation, may be used to predict the impact of altered transport protein function, due to drug interactions, disease states or polymorphisms, on drug disposition

Acknowledgements

William Heizer, MD

Brendan Johnson, PhD

Richard Kowalsky, PharmDGiulia Ghibellini, PhD

Lakshmi Vasist, PharmD

Ann Whitlow

General Clinical Research Center staff

Susan Ford, PharmDSeth Berry, PharmD

Nuclear Medicine & Radiology staff

Jonathan Simpson

National Institutes of Health Grants: GM41935, RR00046GlaxoSmithKline Clinical PK/PD Fellowship Program

Koji Abe, PhDKeith Hoffmaster, PhDXingrong

Liu, PhDHao

Xiong, PhD

Jin Kyung LeeTracy Marion

Imaging Techniques and Pharmacokinetic … L. R. Brouwer School of Pharmacy, University of North...

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