Incorporating In Vitro
Information into Models which
Integrate Intestinal and Renal
Drug Transport
Amin Rostami-Hodjegan, PharmD, PhD, FCP, FJSSX, FAAPS
Professor of Systems Pharmacology
University of Manchester, Manchester, UK
&
Vice President R&D
Simcyp , Sheffield, UK
[email protected]
Drug Transporters: Local vs Systemic PK
Intestine
Lumen
Enterocyte
MDR1 (P-gp)
Blood
MRP
BCRP
Apical
(urine)
PepT1,
PepT2,
OATP1A2,
OATP2B1,
OCT3,
OCTN1,
OCTN2, IBAT,
CNT1, CNT2,
MCT1, MCT4,
MCT5
Liver
OATP1B
1
OATP1B
3
Kidney
Kidney cell
Basolateral
(blood)
OATP1A2
OCTN1
OAT1
OCTN2
OAT2
OAT4
OAT3
URAT1
MATE1
OCT2
MATE2-K
MDR1 (P-gp)
MRP1
MRP2
MRP3
MRP4
MRP6
Blood
Hepatocyte
MDR1 (P-gp)
MRP2
BCRP
The Blood-Brain Barrier
MDR1
Blood
OATP1A2 BCRP
(P-gp)
MRP4
OATP2B1
luminal
OCT1
Endothelial cells
BCRP
abluminal
MRP
3
The Blood-CSF Barrier
BCRP
MRP4
basolateral
Choroid epithelium
apical
MDR1 (P-gp)
Astrocyte feet
Brain parenchyma
Cerebrospinal fluid (CSF)
Scaling from In Vitro Assays
Liver
In vitro data
Jmax/Km or
CLuint T
CLuint, T
per
g Liver
HHEP
In vitro
CLuint, T
SF 1:
REF/RAFHHEP
CLuint, T
per Liver
Intestine
SF 2:
HPGL
SF 3:
Liver Weight
Caco-2, MDCK- II,
LLC-PK1 etc.
Jmax/Km or
CLuint T
CLuint, T
In Jejunum I
Kidney
In vitro data
Jmax/Km or
CLuint T
PTC
In vitro
CLuint, T
SF 1:
REF/RAFPTC
Brain
In vitro data
Jmax/Km or
CLuint T
CLuint, T
per Kidney
SF 1:
REF/RAFJejunum I
Replacement / Additional Organ
SF 2:
PTCPGK
H-BMv
In vitro
CLuint, T
SF 1:
REF/RAFH-BMv
CLuint, T
per
g Kidney
Scaling via the
Permeability and
Surface area
product
SF 3:
Kidney Weight
CLuint, T
per
g Brain
SF 2:
H-BMvPGB
CLuint, T
per Brain
@ BBB
SF 3:
Brain Weight
Jmax/Km or
CLuint T
CLu, T
per whole
organ
User needs to scale to whole organ!
SF: Scaling Factor
Linking Local Sub-Models to Larger PBPK
CYP3A Distribution
Gastric
Emptying
Luminal
Transit
Segregated Blood Flows
Relative P-gp Distribution
Colon
Ileum IV
Ileum III
Ileum II
Ileum I
Jejunum II
Jejunum I
Duodenum
The Advanced Dissolution, Absorption & Metabolism - ADAM
Interplaying Factors: Transporters
Biopharmaceutics & Drug Disposition 34: 2–28 (2013)
fa (ADAM) (Sub)
Effect of REF and Dose on Fa
1.0
0.5
1.0
0.5
0.002 mg
0.0
0.001
0.1
10
1000
0.0
0.001
1.0
400 mg
0.1
2000 mg
0.5
10
1000
0.0
0.001
0.1
10
REF (intestinal P-gp)
As REF ↑ for a P-gp (apical efflux) substrate fa ↓…
1.0
1.0
REF = 0.001
0.5
0.0
0.01
0.1
1
10
100
Dose [mg]
1000 10000 100000
fa (ADAM) (Sub)
fa (ADAM) (Sub)
…effect is less marked as dose ↑
REF = 100
0.5
0.0
0.01
1
100
Dose [mg]
10000
1000
In Vitro-In Vivo Extrapolation of Transporters PK
In vitro apparent active
permeability per gut segment.
Papp,Tran, n 
Peff ,Tran, n
Transfer Papp,Tran,n to in vivo effective active
permeability using the selected prediction model.
Scale up Peff,Tran,n to transport clearance in the
gut segment.
Amount of drug pumped backed into lumen
(efflux) per gut segment.
J max
A  Km  fu gutCent, n 
Peff ,Tran,n Sn FTran, n REFTran, n
CLTran,n fu gutCent, n
In calculations for uptake transporters Clumen is used.
A = area of filter; Sn = Surface area of gut segment; FTrans,n = relative abundance of transporter in gut segment.
Correlations? (e.g. MRP2 in Gut vs. Liver)
 No correlation between the intestinal and hepatic content of MRP2
 Therefore they are independently assigned within Simcyp
 Co-variation of transporters and metabolising enzymes currently under investigation
3
R² = 0.0266
Hepatic MRP2 2
protein
expression
[rel. Units/mg
1
protein]
0
0
1
2
3
4
5
Intestinal MRP2 protein expression [rel. Units/mg protein]
Data from: H. Gläser, thesis 2003
Quantitative Proteomic in Manchester - QconCAT
Schematic of the QconCAT method.
Russell et al 2013, J Proteomics, Revised version under Review
Achour et al, in Preparation
C Y P 4F2
C Y P 3 A4 3
C Y P 3 A7
C Y P 3 A5
C Y P 3 A4
C Y P 2J2
C Y P 2D 6
C Y P 2C 18
C Y P 2C 9
C Y P 2C 8
C Y P 2B 6
C Y P 2 A6
C Y P 1 A2
C y t o c h r o m e P 4 5 0 e n z y m e a b u n d a n c e ( p m o l/m g )
Quantitative Proteomic in Manchester - QconCAT
CYP Abundance and Interindividual Variability
200
B)
150
100
50
0
Achour et al, in Preparation
U G T 2B 15
U G T 2B 7
U G T 2B 4
U G T 1 A9
U G T 1 A6
U G T 1 A4
U G T 1 A3
U G T 1 A1
U G T e n z y m e a b u n d a n c e ( p m o l/m g )
Quantitative Proteomic in Manchester - QconCAT
UGT Abundance and Interindividual Variability
300
B)
200
100
0
Inter-correlations: A Realistic Virtual Patient
Error!
UGT2B15
UGT
2B15
UGT2B7
UGT
2B7
UGT2B4
UGT
2B4
UGT1A9
UGT
1A9
UGT1A6
UGT
1A6
UGT1A4
UGT
1A4
UGT1A3
UGT
1A3
UGT1A1
UGT
1A1
CYP4F2
CYP
4F2
CYP3A43
CYP
3A43
CYP3A7
CYP
3A7
CYP3A5
CYP
3A5
CYP3A4
CYP
3A4
CYP2J2
CYP
2J2
CYP2D6
CYP
2D6
CYP2C18
CYP
2C18
CYP2C9
CYP
2C9
CYP2C8
CYP
2C8
CYP2B6
CYP
2B6
CYP2A6
1
CYP
2A6
CYP
1A2
CYP1A2
0.44
NS
NS
NS
0.45
NS
0.58
NS
NS
NS
0.63
0.47
0.42
0.36
0.48
NS
0.45
0.67
0.70
0.57
1
0.59
0.56
0.68
NS
NS
NS
0.50
NS
NS
0.41
0.35
0.43
NS
0.51
0.38
0.36
0.45
0.59
0.53
1
0.56
NS
NS
NS
NS
0.63
NS
NS
NS
NS
0.35
NS
0.48
NS
NS
NS
0.36
0.49
1
0.56
NS
0.44
NS
0.62
NS
NS
NS
NS
NS
0.42
0.38
NS
NS
NS
NS
NS
1
0.57
NS
NS
NS
NS
NS
0.47
NS
0.43
0.42
0.70
0.48
0.45
0.64
0.66
0.61
1
NS
0.39
NS
NS
-0.43
0.61
0.39
NS
NS
NS
NS
NS
NS
0.43
NS
1
NS
0.37
NS
0.58
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
1
NS
NS
NS
0.42
NS
NS
NS
0.48
NS
NS
0.45
NS
NS
1
0.36*
0.50
NS
NS
0.51
NS
0.48
NS
NS
0.45
NS
NS
1
0.39
NS
0.37
NS
NS
0.43
0.62
0.50
0.43
NS
NS
1
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
1
NS
NS
0.46
0.51
NS
NS
0.51
0.39
NS
1
0.42
NS
NS
0.49
0.45
0.38
0.61
0.46
1
NS
0.41
0.42
NS
0.65
0.50
0.55
1
0.57
0.56
0.55
0.46
NS
NS
1
0.75
0.75
0.82
0.55
0.61
1
0.82
0.72
0.50
0.61
1
0.73
0.61
0.69
1
0.65
0.71
1
0.91
1
Time-variant, Compartmental Luminal Fluid Volumes
Fasted State
250 mL fluid taken with dose:
Average healthy individual
Fasted Gut Lumen Fluid Volumes (250 mL taken with dose)
1000
Whole Gut
Luminal Fluid
Volumes (mL)
Luminal Fluid Volumes (mL)
Stomach
SI Total
100
Duodenum
Jejunum I
II
Colon
10
Ileum I - IV
C bulk(segment ,t) 
Inter-individual
variability not shown
Dissolved mass(segment ,t)
Fluid Volume(segment ,t )
1
0.01
0.1
1
Time (hours)
Time (hours)
10
100
Substrate and Inhibitor with ADAM - DDIs
Luminal Transit
Luminal Transit
Segregated Blood
Flows
Segregated Blood
Flows
CYPs/ Efflux/ Influx Transporters
Note an inhibitor could be an excipient etc
Colon
Ileum IV
Ileum III
Ileum II
Ileum I
Jejunum II
Jejunum I
Duodenum
Colon
Ileum IV
Ileum III
Ileum II
Ileum I
Main inhibitor
Jejunum II
Jejunum I
Duodenum
Substrate
Kidney: Nephron and Its Function
(Guyton & Hall, 2006)
Kidney: Inter-Species Differences and Transporters
Human
Rat
CLrenal
[mL/min/kg]
CLrenal
[mL/min/kg]
CLsecretion
[mL/min/kg]
Famotidine alone
42 ± 9
297 ± 19
196 ± 21
with probenecid
46 ± 10
107 ± 5
22 ± 4
Renal proximal tubule cell
Urine
Octn
Rat
Oat1
(Shitara et al., 2006)
Blood
probenecid
Mrp2,4
Oat3
Oat-K1
No change
Oct1
Oat-K2
famotidine
Oct2
Oatp1
probenecid
Human
Decrease in the
renal clearance
OCTNs
OAT1
MRP2,4
OAT3
OAT4
OCT2
famotidine
Increase in the plasma
concentration
Model Structure: Mechanistic Kidney (Mech KiM)
Glomerulus
Renal mass
Vur-pt1
Henle’s
loop
Distal
tubule
Vur-he
Qur-dt
Vur-dt
Qur-cd1
Collecting
duct
Vur-cd1
Qur-cd2
Vur-cd2
Qblad
Bladder
CLinuc-pt2
Vblad
CLinuc-pt3
PS
Quc-he
uc-he
Qurine
Vce-pt3
Vce-cd1
PS
Quc-cd2
uc-cd2
Vbl-pt2
CLout cb-pt2
CLce-pt2
CLin
PS
Qcb-pt3
cb-pt3
cb-pt3
Vce-dt
PS
Quc-cd1
uc-cd1
Qbl-pt2
Vce-pt2
Vce-he
PS
Quc-dt
uc-dt
Vbl-pt1
CLout cb-pt1
CLce-pt1
CLin
PS
Qcb-pt2
cb-pt2
cb-pt2
Qur-pt3
CLout
PS
Quc-pt3
uc-pt3
uc-pt3
Qur-he
Qbl-pt1
Vce-pt1
CLinuc-pt1
Qur-pt2
CLout
PS
Quc-pt2
uc-pt2
uc-pt2
Vur-pt3
Vbl-glom
CLin
PS
Qcb-pt1
cb-pt1
cb-pt1
uc-pt1
uc-pt1
uc-pt1
Vur-pt2
Qbl-glom=(1-abypass)Qkidney
GFR
Vur-glom
Qur-pt1CLoutPS
Q
Proximal
tubule
Renal blood
Vce-cd2
Qbl-pt3
CLout cb-pt3 Q =b
CLce-pt3
bl-he
bypassQbl-pt
PS
Qcb-he
cb-he
PS
Qcb-dt
cb-dt
Vbl-he
Qbl-cd2
Qbl-pt
Qbl-dt=(1-bbypass)Qbl-pt
Vbl-dt
Qbl-cd1
PS
Qcb-cd1
cb-cd1
PS
Qcb-cd2
cb-cd2
Vbl-pt3
Vbl-cd1
Qbl-vein1
Vbl-cd2
Qbl-vein2
Urine
Qbypass=abypassQkidney
Urinal tubule
Vbl-vein
Qvein
Blood
Modelling Kinetics in the Kidney (Mech KiM)
Filtration
Secretion (passive + active)
Reabsorption (passive + active)
Urinal tubule
Renal blood
Cell (renal mass)
PT – S1
PT – S2
PT – S3
Transporters are available in all
three Proximal Tubule Cell
compartments on the apical and
basal membrane. Thus, the model
can address:
Regional distribution and changes in
activity for transporters as known for
PepT1/PepT2
Nephrotoxicity as well as change in
systemic exposure due to:
HL
DT
• interplay between transporter on
the apical and basal membrane
Cort-CD
Medu-CD
Bladder
• interplay between uptake, efflux
and passive permeation on the
same membrane
• interplay between metabolism and
transporter
Considerations
• GFR directly from the glomerular blood compartment to the glomerular
urinal compartment (filtration);
• Fluid balance within the kidney, i.e. the fluid flow into and out from
the urinal tubular compartments, the renal blood compartments, as
well as the renal mass cell compartments (reabsorption and
secretion);
• Passive permeability at basal and apical sides of each of the renal cell
compartments (passive components of reabsorption and secretion);
• Transporters on basal and apical sides of each of the proximal tubular cell
compartments (active components of reabsorption and secretion);
• Metabolic clearance within the proximal tubular cell compartments;
Input data - Renal In Vitro Models
• Commonly used Renal Cell Lines are MDCK, LLC-PK1, OK, however they are
from animal origin.
• Transfected Cells are used in the industry
- CHO and HEK for the basolateral uptake transporters,
- MDCK and LLC-PK1 for the efflux transporters
• Human Kidney Slices are from intact tissue, however the assay is limited to
basolateral transporters and not a HTS method in the industry (expensive,
technical advanced, tissue demanding)
• Human Proximal Tubule Cells (HPTC):
- HRPT, Caki-1, Caki-2, RPTEC, HK-2, HKC-5
Scaling Factor for Renal Transporters
HEK-293,
CHO etc.
CLuint, T
per
g Kidney
PTC
Jmax/Km
CLuint T
Scaling
Factor 1
In vitro
CLuint, T
Scaling
Factor 2
CLuint, T
per Kidney
Scaling
Factor 3
Input Units
REF/RAFPTC
PTCPGK
Kidney Weight
REF/RAF: Dimensionless factor that reflects the difference in activity and/or
expression between the in vitro system and the in vivo system
Assuming activity of PTC in vitro equals PTC in vivo  REF = 1
PD Inputs from Mech KiM
PD
Basic 2
PD
Basic 1
Output
PD
Link 2
•
PD
Basic 3
Output
PD
Link 3
PD
Basic 3
Output
Output
Output
PD
Link 3
Output
PD will be possible for the cell and the urinal compartments for PT-S1, PT-S2
and PT-S3.
•
Output
The cumulative amount in the cells and urine will be available for PD
simulations (planned for V.12 Release 2).
PBPK to Help with Understanding ‘Local Exposure’
(DDI & Genetic Polymorphism of Transporters)
Xe(t)
X(t)
Compound
PK
C
Effect compartment
E
PD Basic Response
E
Hysteresis
t
AUCtissue 
C
AUCsys .CLin
CLout
Reabsorption of Water – Concentrated Drug 
a
100
Upper proximal tubule
Concentration (mg/L)
Mid proximal tubule
10
Lower proximal tubule
Loop of Henle
1
Distal tubule
Cortical collecting duct
0.1
Medullary collecting duct
0.01
0
48
96
Time (h)
144
Impact on Systemic Drug Concentrations 
Systemic Concentration (mg/L)
b
1
CLPD = 0
CLPD = 0.0001
CLPD = 0.001
0.8
0.6
0.4
0.2
0
0
48
96
Time (h)
144
Impact on Excreted Drug 
Amount of substrate excreted
unchanged in urine (mg)
c
120
100
80
60
40
CLPD = 0
CLPD = 0.0001
CLPD = 0.001
20
0
0
48
Time (h)
96
144
Drug in Plasma, Urine, Kidney Cells 
Impact of Urine pH:
A weak base (pKa 10) (100 mg iv)
80% renally cleared
CLPD = 0.05 ml/min per million cells
120
100
80
60
40
pH 8
pH 7.4
pH 5
20
0
0
24
Time (h)
48
Accumulated Drug
in Urine
14
Systemic Concentration (mg/L)
b
Amount of substrate in Kidney
cells (mg)
Amount of substrate excreted
unchanged in urine (mg)
a
Drug Concentration
in Tubular Cellc 0.5
7
0
0
24
Time (h)
48
0.25
0
0
24
Time (h)
48
Drug Concentration
in Plsma
Memantine – CLrenal pH-dependency
Plasma Concentration (µg/L)
Memantine
primary amine
pKa = 10.27
logP = 3.2
95% renally cleared
Acidic Urine (pH 5)
Alkaline Urine (pH 8)
45
45
40
40
35
35
30
30
25
25
20
20
0
4
• CLPD: 50 µl/min/million cells (PE
from urine pH 7)
• OCT2 (basal uptake):
4.7µl/min/million cells
• ASSUMED apical efflux in the
same range!
Burt et al., 2012 Gordon Conference
8
12
Time (h)
16
20
24
Acidic Urine (pH 5)
0
4
8
12
Time (h)
16
20
Alkaline Urine (pH 8)
24
Transporter Inhibition/Induction/Genetics
50
40
30
Uptake CL
20
10
b
0.002
Systemic Drug C(t)
0.001
0.0005
0
0
6
12
18
24
Time (h)
0.01
0.008
Urine Drug C(t)
0.006
0.004
0.002
0
0
d
Concentration in the upper proximal
tubule cell compartment (mg/L)
Concentration in the upper proximal
tubule urine compartment (mg/L)
Inhibition of Major Route
0.0015
0
c
No Inhibition
Inhibition of Minor Route
6
12
18
24
e
Time (h)
0.01
0.008
Tubular Drug C(t)
0.006
0.004
0.002
6
12
Time (h)
18
24
0.01
0.008
Efferent Blood C(t)
0.006
0.004
0.002
0
0
0
Concentration in the upper proximal
tubule blood compartment (mg/L)
Intrinsic uptake clearance on
basolateral interface (L/h)
a
0.0025
Systemic Concentration (mg/L)
Transporter Inhibition/Induction/Genetics
0
6
12
Time (h)
18
24
0
6
12
Time (h)
18
24
Concentration in medullary
collecting duct compartment (mg/L)
Physiological Variability
80
60
40
20
0
0
12
Time (h)
24
Further Details
Springer Book Chapter: Bente Steffansen & Yuichi Sugiyama; Editors
Howard Burt
Linzhong Li
Gaohua Lu
Sibylle Neuhoff
[email protected]
[email protected]
[email protected]
[email protected]

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