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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?
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)
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
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
Liver 2 Liver 3 Liver 4Liver 1
Acc
umul
atio
n (p
mol
/mg
prot
ein)
BEI=50% BEI=55%
BEI=56%
BEI=66%
Liver 2 Liver 3 Liver 4Liver 1
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%
Liver 2 Liver 3 Liver 4Liver 1
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%
Liver 2 Liver 3 Liver 4Liver 1
Acc
umul
atio
n (%
dos
e/ m
g pr
otei
n)
BEI=34%
BEI=20%
BEI=31%
BEI=54%
Liver 2 Liver 3 Liver 4Liver 1
Acc
umul
atio
n (%
dos
e/ m
g pr
otei
n)99mTc-Mebrofenin
0.0
0.1
0.2
0.3
0.4
Liver 2 Liver 3 Liver 4Liver 1
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
Liver 2 Liver 3 Liver 4Liver 1
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%
Liver 2 Liver 3 Liver 4Liver 1
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%
Liver 2 Liver 3 Liver 4Liver 1
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
Xiong, H. et al., J Pharmacol Exp Ther 295:512-518, 2000
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
Xiong, H. et al., J Pharmacol Exp Ther 295:512-518, 2000
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
Xiong, H. et al., J Pharmacol Exp Ther 295:512-518, 2000
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