In vitro - in vivo Extrapolation toxischer
Dosen: Das „equivalent exposure“ Konzept
Michael Gülden
Institut für Toxikologie und Pharmakologie für Naturwissenschaftler
Universitäts Klinik Schleswig-Holstein
Campus Kiel, Brunswiker Str. 10, D-24105 Kiel, Germany
Quantitative in vitro – in vivo Extrapolation
In vitro
Toxische
Konzentrationen
?
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
In vivo
Externe
Dosis/Exposition
2
Keine einfachen Lösungen des Problems
W. Halle (2003) The Registry of Cytotoxicity: Toxicity Testing in Cell Cultures to Predict
Acute Toxicity (LD50) and to Reduce Testing in Animals. ATLA 31, 89-198
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
3
Quantitative in vitro – in vivo Extrapolation
In vitro
Toxische
Konzentrationen
?
In vivo
Externe
Dosis/Exposition
!
“Pharmakokinetik”
in vitro
Pharmakokinetik
• Resorption
• Verteilung
• Metabolismus
• Exkretion
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
4
In vitro Systeme
Principal components:
• Cells
• Culture medium
• Culture vessel
Serum ≤ 20%
Relative cell volume « 1% of total volume
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
5
In vitro bestimmte Konzentrations-Wirkungsbeziehungen
basieren auf nominellen Konzentrationen
Principal components:
100
• Cells
• Culture vessel
% viable cells
• Culture medium
50
Serum ≤ 20%
EC 50
0
Relative cell volume « 1% of total volume
10 1
10 2
Concentration (µM)
Nominelle Konzentrationen
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
6
Verteilung und Elimination in vitro
Nominelle effektive
Konzentration (EC50)
• Serumbindung
• Zellbindung
• Adsorption an
Kulturgefäßmaterial
• Metabolisierung
• Dekomposition
Freie effektive
Konzentration (ECu50)
• Evaporation
Toxizität
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
7
Binding of drugs to microsomes
Human liver microsomes
Warfarin
Propranolol
Imipramine
(Obach R.S., Drug Metab. Dispos.1997, 25, 1359-1369)
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
8
Binding of drugs to hepatocytes
(3 µM, 106 cells/ml)
Verapamil
Propranolol
Oxaprozin
Omeprazole
Metyrapone
Metoprolol
Isradipin
Idapamide
Glyburide
Diazepam
Clozapine
Cerivastatin
Bumetanide
Betaxolol
Astemizole
Albendazole
2-Ethoxybenzamide
0.0
0.5
1.0
Fraction unbound (fu)
(Austin et al., Drug Metab Dispos 2005, 33 (3), 419-425)
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
9
Binding of octylphenol to serum
Estrogenicity reporter gene assay using a human cell line
EC 50,nominal (µM)
30
EC 50,nominal
EC 50,free
20
10
0
0
A
10
20
30
40
50
60
EC 50,free (µM)
% fetal calf serum
0.05
0.00
0
10
20
30
40
50
60
% fetal bovine serum
(Heringa et al., Environ Sci & Technol. 2004, 38, 6263-6270)
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
10
Influence of albumin concentration on the cytotoxic
potency of pentachlorophenol in proliferating Balb/c 3T3 cells
% of control
100
EC50 = 32 µM
EC50 = 1400 µM
50
5% FCS
5%FCS +1 %BSA
5%FCS +2% BSA
5%FCS +4 % BSA
0
10 1
10 2
PCP concentration (µM)
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
10 3
11
Influence of albumin concentration on the cytotoxic
potency of pentachlorophenol in proliferating Balb/c 3T3 cells
EC 50 (µM)
100
% of control
Specific binding (moles/mol)
1500
50
5% FCS
EC50 = 34.5 + 2.3 x [BSA]
1000
500
5%FCS +1 %BSA
5%FCS +2% BSA
5%FCS +4 % BSA
0
0
10 1
10 2
PCP concentration (µM)
10 3
0
100
200
300
400
500
600
Albumin concentration added (µM)
(Gülden et al., 2002. Toxicology 175, 201-213
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
12
Influence of cell concentration on the cytotoxic
potency of methylmercury in bull sperm cells
ATP content (% of control)
125
100
EC50 = 23 µM
75
EC50 = 2.9 µM
50
15 x 106 cells/ml
30 x 106 cells/ml
25
60 x 106 cells/ml
120 x 106 cells/ml
0
0.1
1
10
100
MeHg concentration (µM)
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
13
125
25
100
20
EC 50 value (µM)
ATP content (% of control)
Influence of cell concentration on the cytotoxic
potency of methylmercury in bull sperm cells
75
50
15 x 10 6 cells/ml
6
30 x 10 cells/ml
25
60 x 10 6 cells/ml
0.2 nmol/106 cells
15
10
5
120 x 10 6 cells/ml
0
0.1
1
10
100
MeHg concentration (µM)
0
0
10
20
30
40
50
60
70
80
90
100
110
120
6
Cell concentration (10 /ml)
(Gülden et al., 2001. Toxicol. in Vitro 15, 233-243)
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
14
130
Quantitative in vitro – in vivo Extrapolation
Das „equivalent exposure“ Konzept
In vitro
In vivo
Nominelle effektive
Konzentration (EC50)
Externe
Dosis/Exposition
Nominelle Konzentration,Gewebedosis
Freie effektive
Konzentration (ECu50)
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
Freie
Gewebekonzentration
15
Distribution model for chemicals in vitro
extracellular medium
n•P
Cb = ————— = B • P
1 + KD/Cu
bound to serum
albumin
(Cb)
Lipid
cells
freely
dissolved
(Cu)
freely
dissolved
(Cu)
(VL,e)
partitioned
into serum
lipids
(CL,e)
CL = Kow • Cu
Lipid
partitioned
into cell lipids
(CL,c)
(VL,c)
B: Specific binding (moles/mole)
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
16
Distribution equation
System specific
factors
Relative lipid
volume in the in
vitro system
Albumin concentration in the
medium
EC50 = ECu50(1 + Kow•V´L) + B•P
Substance
specific factors
Octanol/water
partition coefficient
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
Specific binding
to albumin
17
Bioavailability of chemicals in an in vitro system
Example: Balb/c 3T3 test system
Composition
Cell concentration at seeding:
3x
104/ml
Albumin concentration in the
culture medium (5% FBS):
18 µM
Relative lipid volume:
free fraction (fu)
1,0
0,8
Phenol
●
● ●
MCP
0,6
0,4
●
●
4-NP ●
4-OP
fra 0,00,8 PCP
to cti
al on 0,6
bu b 0,4
m ou
in n 0,2
(f d
b)
0.1 µl/ml
●
TCP●
0,2
DCP
0,0
1
2
Lindan
● Dieldrin
DDT●
3
l og
4
5
6
7
K ow
(Gülden & Seibert, 2005. Aquatic Toxicology 72, 327-337)
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
18
p,
H
p´
e
Pe xa p, -DD
nt chl p´- E
ac or D
h o D
4- lor phe T
N o n
2,
4, 4 ony phe e
5- -O lp n
Tr c h ol
ic tyl en
hl ph o
or e l
op no
h l
Th Di eno
io eld l
Tr
rid ri
ib
a n
ut
L
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M A ch an
2, erc mit lor e
4- ur rip id
D ic t y e
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4- hlo hlo ne
C ro rid
hl ph e
or e
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M
2,
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ta init ar ion
ss ro fa
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D
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Pr ph cid
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n
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ra olo
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r
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Et m eta at
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ox
Is
Ph ide
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l a an
Li
th M lco ol
iu e h o
m t
Ph s han l
en ulp ol
ob ha
ar te
bi
ta
l
Fraction unbound (fu)
Bioavailability of chemicals in an in vitro system
Example: Balb/c 3T3 test system
1.0
0.5
0.007
0.001
0.0
(Gülden & Seibert, 2007. ATLA 35, 39-46)
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
19
Application of the distribution model to estimate free
cytotoxic concentrations
Nominal cytotoxic concentration in vitro (EC50)
EC50 much higher than
albumin concentration
(EC50 » P) ?
no
yes
Determine specific binding to
albumin (B)
Lipophilicity below
critical value
(Kow• V´L < 1) ?
yes
no
Lipophilicity below
critical value
(Kow• V´L < 1) ?
no
yes
EC50- B•P
----------------1 + Kow• V´L
EC50- B•P
EC50
----------------1 + Kow• V´L
EC50
Free cytotoxic concentration in vitro (ECu50)
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
20
„Equivalent exposure“ Konzept
Anwendungsbeispiel
Verwendung von in vitro
Zytotoxizitätsdaten zur Vorhersage der
akuten Toxizität für Fische
Fish acute toxic potency vs. cytotoxic potency
Fish acute toxicity log LC50 (M)
1
LC50
0
-1
• Endpoint: death
-2
• Free aquatic concentrations
-3
(OECD guideline test)
-4
-5
EC50
-6
• Endpoint: cell death
-7
-7
-6
-5
-4
-3
-2
-1
0
1
Fish cell line R1 cytotoxicity log EC50 (M)
• Nominal concentrations
• Medium with 10% FBS
Segner H. & Lenz D., 1993. Cytotoxicity assays with the rainbow trout R1 cell
line. Toxicology in Vitro 7, 537-540
Segner H., 2004. Cytotoxicity assay with fish cells as an alternative to the acute
lethality assay with fish. ATLA 32, 375-382
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
22
Fish acute toxicity log LC50 (M)
Improvement of in vitro toxicity testing for aquatic toxicity
assessment
Fish cell line: R1
Mammalian cell line: Balb/c 3T3
• cell death
• 24 hours exposure
• nominal EC50-values
• cell growth inhibition
• 72 hours exposure
• nominal EC50-values
• free ECu50-values
1
1
1
0
0
0
-1
-1
-1
-2
-2
-2
-3
-3
-3
-4
-4
-4
-5
-5
-5
-6
-6
-6
-7
-7
-7
-8
-8
-7
-6
-5
-4
-3
-2
-1
0
R1 fish cell line cytotoxicity (24h) log EC50 (M)
1
-8
-8
-7 -6 -5 -4 -3 -2 -1
0
Balb/c 3T3 cytotoxicity (72h) log EC50 (M)
1
-8
-8
-7
-6
-5
-4
-3
-2
-1
0
1
Balb/c 3T3 cytotoxicity (72h) log ECu50 (M)
Segner H., 2004. ATLA 32, 375-382
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
23
10 7
10 7
10 6
10 6
10 5
10 5
10 4
Fish LC50 (µM)
Rainbow trout
Fish LC50 (µM)
Improvement of in vitro toxicity testing for aquatic toxicity
assessment
10 3
10 2
10 1
10 4
10 3
10 2
10 0
10 0
10 -1
10 -1
10 -2
10 -2
(Gülden & Seibert, 2005.
Aquatic Toxicology 72,
327-337)
10 7
10 7
10 6
10 6
10 5
10 5
10 3
10 2
10 1
Lindane
Balb/c 3T3 ECu50 (µM)
Fish LC50 (µM)
Fish LC50 (µM)
Balb/c 3T3 EC50 (µM)
Fathead minnow
Malathion
Dieldrin
10 -3
10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 10 6 10 7
10 -3
10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 10 6 10 7
10 4
Nicotine
10 1
10 4
10 3
10 2
Nicotine
10 1
10 0
10 0
10 -1
10 -1
10 -2
10 -2
10 -3
10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 10 6 10 7
10 -3
10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 10 6 10 7
Balb/c 3T3 EC50 (µM)
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
Lindane
Dieldrin
Balb/c 3T3 ECu50 (µM)
24
Quantitative in vitro – in vivo Extrapolation
Das „equivalent exposure“ Konzept
In vitro
In vivo
Balb/c 3T3-System
Nominelle zytotoxische
Konzentration (EC50)
HumanserumKonzentration
Freie zytotoxische
Konzentration (ECu50)
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
Freie
Gewebekonzentration
26
Differences in composition of human serum and in vitro
systems
Balb/c 3T3 cell
system
Human serum
Ratio
Albumin
concentration
18 µM
600 µM
33
Lipid volume
fraction
0.1 ml/l
6 ml/l
60
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
27
Prediction model for equivalent human serum concentrations
70
EC serum/EC 50,invitro
60
DDT
50
PCP
4-OP
●
40
TCP
30
vi
tr
o
●
●
4-NP
● Dieldrin
●
20
10
0
0.8
fb 0.6
in
●
● Lindan
● DCP
● MCP
0.4
0.2
Phenol
0.0
●
1
2
3
log
4
5
6
7
K ow
1 + Kow• V´L,serum
ECserum = (EC50-B•Pinvitro) ———————— + B•Pserum
1 + Kow• V´L,invitro
(Gülden et al. 2003, Toxicology 189, 211-222)
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
28
p,
ex
p
ac p -D
D
,
4- hlo p-D T
r
N o D
on ph E
yl en
ph e
Pe 4
e
nt -Oc Di no
ac t y e l d l
2,
hl lp ri
4,
5- T oro hen n
Tr hi ph o
ic or en l
hl id o
or az l
op in
2,
h e
4- A L i e n
D m nd ol
ic itr a
hl yp ne
o r ti
op lin
4h e
C
hl H eno
or g l
o C
M phe l2
al n
D
at ol
ex
hi
W
tro
o
a
pr
rfa n
op
r
ox 2 , in
4
y
Ac
p h -D
et
en
yl V
ic e K e
sa ra C
lic p N
Pr ylic ami
op a l
ra cid
Ph
n
en Ph olo
ob en l
Et
a o
hy L rbi l
le i2 S tal
ne O
gl 4
Is A yco
op s l
ro 2 O
p 3
Tl ano
M 2 SO l
et
h 4
Pa E an
ra tha ol
ce n
t o
Th N am l
eo ico ol
p h tin
Pa yll e
ra ine
qu
at
H
Ratio Cserum/EC50
Human serum concentrations equivalent to EC50-values in Balb/c 3T3
cell cultures
60
50
40
30
20
10
0
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
29
Relative potency (potency ranking) in vivo may be
completely different compared to in vitro
1000
1000
1000
500
500
500
0
0
0
Pe
nt
al
liu
m
Th
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
Pr
D
ex
op
tro
ra
no
pr
lo
op
l
o
Pe
xy
ph
nt
ac
en
hl
e
or
op
he
no
l
1500
ac
hl
or
op
he
4C
no
hl
l
or
op
he
no
l
Ph
en
ol
1500
te
M
al
at
hi
on
Li
nd
an
e
1500
su
lfa
Concentration (µM)
EC50 (µM)
ECserum (µM)
30
Strategy for extrapolation of equivalent serum concentrations
Nominal toxic concentration in vitro (EC50)
EC50 higher than serum
albumin concentration
(EC50 > 600 µM) ?
no
yes
Determine specific binding to
albumin (B)
Lipophilicity below
critical value
(Kow ≤ 102)?
Lipophilicity below
critical value
(Kow ≤ 102) ?
yes
yes
no
no
1 + Kow• V´L,serum
(EC50-B•Pinvitro) ----------------------- + B•Pserum
1 + Kow• V´L,invitro
EC50+B(Pserum-Pinvitro)
1 + Kow• V´L,serum
EC50 ----------------------1 + Kow• V´L,invitro
EC50
Equivalent concentration in serum
(Gülden et al., 2006. Toxicol. in Vitro 20, 1114-1124)
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
31
Quantitative in vitro – in vivo Extrapolation
Das „equivalent exposure“ Konzept
In vitro
In vivo
Nominelle effektive
Konzentration (EC50)
Externe
Dosis/Exposition
Serumbindung
Zellbindung
Adsorption
Metabolismus
Dekomposition
Evaporation
Resorption
Verteilung
Metabolismus
Exkretion
Nominelle Konzentration,Gewebedosis
Verteilung
Freie effektive
Konzentration (ECu50)
M.Gülden, DGPT/BfR-workshop “Dosismetrie in vitro - in vivo”, Berlin, 14.10.2008
Freie
Gewebekonzentration
32