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K.L.R. BrouwerPrinciples of Toxicokinetics/Toxicodynamics
TOXC 442
1
Biochemical and Molecular ToxicologyENVR 442/TOXC 442/BIOC 442
Principles of Toxicokinetics/Toxicodynanics
Kim L.R. Brouwer, PharmD, PhD
[email protected]; 919-962-7030
Pharmacokinetics/
Toxicokinetics:
the study of the time course of
xenobiotic absorption,
distribution, metabolism and
excretion
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ADME
Absorption
– how the xenobiotic enters the body
Distribution
– where the xenobiotic goes in the body
Metabolism
– what the body does to the xenobiotic
Elimination
– how the xenobiotic is removed from the body
Pharmacodynamics/
Toxicodynamics:
the relationship between
xenobiotic concentration at the
site of action and the resulting
effect, including the time course
and intensity of therapeutic and
adverse effects
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Pharmacokinetics/ Pharmacodynamics/
Toxicokinetics Toxicodynamics
Dosage
Regimen/
Exposure
Plasma
Concen-
tration
Site
of
Action
Effects:Pharmacologic
Toxic
Toxicokinetic/ADME Studies
Pharmacokinetics / Bioavailability
Mass Balance
Tissue Distribution
Metabolite Profile
Plasma Protein Binding
Inhibition / Induction
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Clinical TestingPre-Clinical
Testing
In Vitro
PK / PD
Animal
PK / PD
Toxicology
PK-Guided
Dose
Escalation
Safety
Assessment
Phase 1
Dose/Response
Efficacy
Dose Selection
Patient
Variables
Pop’n PK
In Large
Efficacy
Trials
Special
Populations
Phase 2
Phase 3
Bio-
Equivalence
Generics
Product line
extension
Post
Marketing
Terms used in
Pharmacokinetics/Toxicokinetics:
Cmax
Tmax
t 1/2
AUC
AUMC
Vdss
fu
Cl
MRT
MAT
F
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Cmax
maximum concentration of compound
observed in the matrix of interest
Tmax
time of maximum concentration
100
1000
10000
0 5 10 15 20 25
Cmax
Time
Conce
ntr
atio
n
Tmax - time of maximum concentration
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lambda ()
terminal elimination rate constant
(slope from a semi-log concentration vs time plot)
t 1/2
half-life: the time it takes for the
concentration of the compound to
decrease by 50%
100
1000
10000
0 5 10 15 20 25
- slope =
t12
2
ln
Time
Conce
ntr
atio
n
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The Half-life is
Compound- and Subject-Dependent
Compound Name t1/2 (h)
Rosiglitazone Avandia 3-4
Lamotrigine Lamictal 25-33
Buproprion Wellbutrin 21
Fluticasone Flovent n/a
Paroxetine Paxil 21
Data from LexiComp
Half-life
Half-life is used to:
– Determine the time to reach steady-state (5 x t1/2)
– Determine the time to eliminate all the compound from
the body (5 t1/2)
– Calculate dosing intervals
– Determine how much compound accumulates in the body
given a fixed dosing interval or exposure
Half-life is a secondary pharmacokinetic parameter
that is determined by the volume of distribution (V)
and clearance (Cl) of the compound
Cl
xVt
693.0
21
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AUC
area under the
concentration
vs time curve
ln C
on
cen
trati
on
AUC
Time
tau
ln C
once
ntr
atio
n
Time
AUC 0 = AUC tau
Single Dose Multiple Dose to Steady-State
Time
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Steady-state
At Steady-State:
Rate of input = Rate of elimination
AUMC
area under the
first moment
curve
Tim
e * C
on
cen
trati
on
AUMC
Time
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Mean Residence Time (MRT)
The average time one molecule resides in
the body
MRT = AUMC/AUC
Used to express overall persistence of
compound in the body
Clearance (Cl)
– volume of fluid (usually blood) from which compound is removed completely per unit time
Mathematically:
ClDose
AUC
Organs that may be involved in clearance:» GI tract
» Liver
» Kidney
» Lungs
» Other sites (e.g., blood, skin)
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Clearances (Cl) are Additive
Pharmacokinetic Parameters of
GI158104X in Dog
Dosemg/kg/day
ClIV(mL/min/kg)
Clr(mL/min/kg)
9.5 10 0.7
Clother(mL/min/kg)
9.3
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Linear Pharmacokinetics
Linear Kinetics = Dose-Proportional kinetics
AUC (or concentration) changes
proportionally with dose
DoseA
UC
Avandia Package Insert
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Stationary Pharmacokinetics
Pharmacokinetic parameters are
independent of time
Package insert for Tegretol (carbamazepine)
Because Tegretol induces its own metabolism, the half-life is also
variable. Autoinduction is completed after 3-5 weeks of a fixed dosing
regimen. Initial half-life values range from 25-65 hrs, decreasing to
12-17 hr s on repeated doses. Tegretol is metabolized in the liver.
Volume of Distribution
at Steady-State (Vdss)
a parameter that relates
plasma concentration to
total mass of compound in
the body
Mathematically:
VdDose AUMC
AUCss *
2
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The Volume of Distribution is
Compound- and Subject-Dependent
Compound Name V (L)
Rosiglitazone Avandia 18
Lamotrigine Lamictal 70
Buproprion Wellbutrin 140
Fluticasone Flovent 280
Paroxetine Paxil 560
Data from LexiComp
Fraction Unbound (fu)
– fraction of drug that is not
bound to plasma proteins
– the unbound
concentration is in
equilibrium between the
tissues and blood
Blood
Tissue
Bound
Drug
Unbound
Drug
Bound
Drug
Unbound
Drug
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Protein-Binding Changes May be Clinically
Relevant for the Following Types of
Compounds and Routes of AdministrationHigh Extraction
Ratio
Low Extraction
Ratio
IV Administration
Hepatic Clearance Yes No
Nonhepatic Clearance Yes No
Oral Administration
Hepatic Clearance No No
Nonhepatic Clearance Yes† No
† No drugs from a list of 456 met these criteria
Benet and Hoener, Clin Pharmacol Ther 71:115, 2002
Organ Extraction (E)
Clearing
OrganCin Cout
Q Q
Drug Elimination
E =Cin - Cout
Cin
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When Is Protein Binding
Important?
In vitro ADME/preclinical toxicology studies
Scaling pharmacokinetic/pharmacodynamic parameters from animal models to humans
Calculating human doses from in vitromeasures of target concentrations
Therapeutic drug monitoring of blood/plasma concentrations for drugs with a narrow therapeutic index
Bioavailability (F)
fraction of the administered
dose that reaches the systemic
circulation intact
0 < F < 1
Mathematically:
FAUC
AUC
Dose
Dose
PO
IV
IV
PO
*
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Relative Bioavailability
fraction of the dose of a test product
that reaches the systemic circulation
relative to a reference product
Mathematically:
FAUC
testAUC
ref
Doseref
Dosetest
*
Bioavailability is an important
determinant of pharmacologic
response and therapeutic
effectiveness
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Estimation of Oral Bioavailability (F)
After an IV and Oral Dose of
Compound X
Dosemg/kg/day
AUCIV1
(g*hr/mL)
AUCPO1
(g*hr/mL)
5.0 6.25 5.94
25.0 32.4
1 mean, n=4
8.15
F
0.25
0.95
Factors Responsible for Poor
or Variable Oral Bioavailability
Physicochemical Factors
Physiologic Factors
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Influence of Formulation Factors
on the Concentration-Time Profile
Formulations with
different release rates
(absorption kinetics)
Time, h
0 4 8 12 16 20 24 28 32 36Ro
sig
lita
zo
ne P
lasm
a C
on
cen
trati
on
, n
g/m
L
0
50
100
150
200
CC CCCC
C
C
C
C
C
C
C
C
C
CDDDDD
D
D
D
D
D
D
D
D
D
D
D
EE
EE
E
E
E
E
E
E
E
E
E
E
E
E
EFFF
F
F
F
F F F
F
FF
F
F
F
F
GG
G
G
G
G
G
G
GG
G
G
G
G
G
G
H
H
H
H
H
H
HH H
H
H
H
H
H
H
H
Formulation #1CFormulation #2DFormulation #3EFormulation #4FFormulation #5GFormulation #6H
Influence of Formulation on the
Bioavailability of a Compound
Bioequivalence
studies are
performed to select
the formulation
which gives the
greatest oral
exposure to the drug
Serum Concentration vs Time Profile
0
50
100
150
200
250
0 5 10 15
Time (hours)
Co
nc
en
tra
tio
n (
ng
/mL
)
Form. A
Form. B
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Special Studies:
Effects of Food on Exposure
Often the fed/fasted
state of the animal can
influence the
absorption of drugs
Food can enhance or
inhibit absorption
Can impact on clinical
dosing0
500
1000
1500
2000
2500
3000
3500
4000
4500
Fed Fasted
AUC (inf)(ng*hr/mL)
Physiologic Factors Responsible
for Poor or Variable Oral
Bioavailability
Cell Membranes
pH, Volume and Content of GI Fluid
GI Motility
Vascularity and Blood Flow
Enzymatic Degradation / Metabolism
Disease States
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The Gastrointestinal Tract
Presystemic Elimination: (First-Pass Effect)Loss or metabolism of drug between the
administration site and the sampling site
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Liver
Portal VeinGut
Lumen
Gut
Wall
Systemic
Circulation
Metabolism
Feces
Factors Affecting Systemic Availability
Solubility
Chemical Stability
Permeability
Mucosal Metabolism
Transport/Efflux
Protein Binding
RBC Partitioning
Blood/Plasma Stability
Metabolism
Biliary Excretion
First-pass Intestinal and Hepatic
Metabolism of Cyclosporin
Benet et al., J Controlled Release 39:139, 1996
14%51% 8%
27%
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In Vivo Pharmacokinetic
and Bioavailability Studies Resulting Information:
– pharmacokinetic parameters (t1/2, AUC, Vdss,
Cl, F) in the species used for the multiple dose
toxicology studies
– rate and extent of drug absorption
– primary routes of elimination
Utility:
– identifies bioavailability problems
– parameters are used in scaling to humans to
predict the initial human dose
Additional Studies for Compounds
That Exhibit Poor Bioavailability
In Vivo Studies to Assess Sites of
Presystemic Elimination:
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Liver
Portal Vein
Systemic
Circulation
Assessing Sites of Presystemic
Elimination
Duodenum
Jejunum
& Ileum
Dose
DoseStomach
Dose
Additional Studies for Compounds
That Exhibit Poor Bioavailability
In Vivo Portal Vein Administration Studies:
– If FPV approximates 100%, presystemic
elimination occurs prior to the portal vein (gut
lumen or gut wall)
– If FPV approximates FPO, presystemic
elimination occurs primarily in the liver
PV
IV
IV
PVPV Dose
DoseAUCAUCF *
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Liver
Portal Vein
Systemic
Circulation
Assessing Sites of Presystemic
Elimination
Duodenum
Jejunum
& Ileum
Dose
DoseStomach
Dose
Dose
Case Study: Assessing Sites of
Presystemic Elimination
Route of
Administration
AUC
(g*hr/ml)
Bioavailability
(%)
Oral 972 21
Intraduodenal 1378 30
Portal Vein 4386 94
Intravenous 4653 100
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In Situ and In Vitro Models to Assess
Sites of Presystemic Elimination
Models to Assess Presystemic Elimination Prior to
the Portal Vein
– Isolated Perfused Intestinal Segments
– Everted Gut Sacs
– Intestinal Rings
– Cell Monolayers
Models to Assess Hepatic Presystemic
Elimination
– Isolated Perfused Liver
– Liver Slices
– Hepatocytes (freshly isolated/cultured)
Toxicokinetics
Dose Selection
Dose Proportionality
Multiple Dose Pharmacokinetics
Induction/Inhibition
Exposure Verification
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Toxicokinetics: Dose Selection
Determination of the optimum dose
– primarily focused on the relationship between
dose and exposure
» linearity
» saturation
Cmax and AUC Following Escalating
Doses of Compound A
Dosemg/kg/day
Cmax 1
(ug/mL)
AUC 1
(ug*hr/mL)
5.0 1.40 6.25
25.0 9.47 32.4
100.0 10.56 47.2
1 mean, n=4
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Dose Selection
0
2000
4000
6000
8000
10000
12000
0 100 200 300 400
Dose (mg/kg/day)
AUC
(ng*h/mL)
Rat
Dog
Saturation of Absorption
0
5
10
15
20
25
30
35
40
25 500 10 500
Dose (mg/kg)
Bio
avail
ab
ilit
y (
%)
Mouse Dog
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Toxicokinetics: Dose Proportionality
A = AUC increases
proportional to dose.
B = Less than
proportional increase in
exposure: Absorption-
limited exposure.
C = Greater than
proportional increase in
exposure: Capacity-
limited elimination0
200
400
600
800
1000
1200
1400
1600
0 50 100 150 200 250 300
Dose (mg/kg)
AU
C h
r*µ
g/m
L
A
B
C
Interpretation of results demonstrating lower toxicity at higher doses
depends on whether compound exhibits dose-proportional kinetics.
Toxicokinetics:
Multiple Dose Pharmacokinetics Determine how drug distribution is altered after
multiple dosing
– Examine changes in clearance, half-life, accumulation,
linearity
– Assure dose related continuous exposure of the animals
to the test compound
Evaluate:
– Toxicokinetic changes with dose and time
– Effect of advancing age of the animals
– Decreases in lean body mass, total body water, cardiac
output, renal, hepatic and GI tract blood flow
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Terminal t1/2 (mean and SD) on
Days 1 and 30 During a Rat 1-Month
Oral Multiple Dosing Study
Males Females
Half-life (hr) Half-life (hr)
Dose Day 1 Day 30 Dose Day 1 Day 30
(mg/kg/day) (mg/kg/day)
5 2.94 2.31 5 2.15 1.72
0.86 0.29 0.10 0.09
25 2.18 2.06 25 2.45 1.99
0.30 0.25 1.07 0.39
150 15.33 2.14 150 9.76 2.72
3.32 0.14 4.54 0.71
Effect of Age on the Toxicokinetics of
N-Acetylprocainamide HCl After IV
Administration of 100 mg/kg in RatsAge
Parameters
Half-life(hr)
C (0)(ug/mL)
AUC(ug*hr/mL)
Cl(L/hr/kg)
Vd(L/kg)
13 WeeksMean
1.66
19.0
45.3
2.01
4.75
(n=7)CV%
12.1
12.1
18.2
14.8
11.2
26 WeeksMean
1.82
27.7
72.6
1.29
3.35
(n=11)CV%
14.2
21.6
27.0
23.5
22.0
52 WeeksMean
2.29
46.9
156.0
0.664
1.98
(n=7)CV%
42.3
21.8
51.4
71.4
24.9
From: S. Garattini, Drug Metabolism Reviews 13, (3), 1982
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Time (Exposure) Dependence
0
5
10
15
20
25
30
35
40
0 5 10 15 20 25
Time
Inhibit ion
Normal
Induct ion
1
10
100
0 5 10 15 20 25
Time
Inhibit ion
Normal
Induct ion
Induction and Inhibition of
metabolism as examples of
elimination changes
Induction or Inhibition
may occur without
affecting the overall
disposition of the test
compound
Exposure Verification -example
Continuous IV infusion for 14 days in rats
and dogs
Infusion solution at pH = 5
Blood samples taken at various times to
document exposure
Pilot (24 hr) study in both species
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Exposure Verification
1
10
100
1000
10000
0 50 100 150 200
Con
cen
trati
on
(n
g/m
L)
Time (h)
Males
Females
Expected
Special Toxicokinetic Issues
Toxicity / Potency Evaluations
Metabolic Sites
Correlation of Residual Drug with Toxicity
Species Selection
Gender Effects
Placental Transfer
Milk Transfer
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Coverage for
Human Metabolites
From: Ferrero, J.L. and Brendel, K. Advances in
Pharmacology V43, pp131-169, 1997.
Species Selection
confirmation that the
species chosen for
oncogenicity studies
supports the human
metabolism
is there coverage in the
animal species for all
metabolites generated in
human systems
Cumulative
Percent of
Radioactive Dose
in Urine and
Feces of Male
Cynomolgus
Monkeys After a
Single IV Dose of
[14C]GW280430
Collection Percent of Radioactive Dose
Interval Animal Number
(Hours) I04646 I04647 I04650 I04651 I04652 I04653 Mean SD
Urine
0-2 14.5 4.16 25.1 20.3 0.01 ND 10.7 10.80-6 42.6 31.2 40.4 34.0 32.3 10.0 31.8 11.6
0-12 43.3 35.2 45.0 38.6 34.2 10.0 34.4 12.70-24 46.2 37.7 49.1 39.7 35.7 27.8 39.4 7.640-48 48.1 40.0 51.0 42.7 37.4 32.7 42.0 6.780-72 49.2 41.0 51.8 44.1 38.6 35.1 43.3 6.360-96 50.0 42.1 52.4 45.1 39.6 36.3 44.2 6.160-120 50.8 42.9 52.9 45.8 40.4 37.1 45.0 6.050-144 51.9 43.6 53.1 46.5 41.4 37.6 45.7 6.030-168 52.3 43.9 53.5 46.7 41.8 37.9 46.0 6.05
Feces
0-24 15.5 20.5 10.7 7.78 20.8 12.8 14.7 5.290-48 22.2 31.0 28.4 30.8 26.1 30.1 28.1 3.410-72 23.6 32.8 31.2 36.3 28.5 33.2 30.9 4.410-96 23.9 33.4 31.6 37.0 29.2 33.9 31.5 4.510-120 30.6 33.8 32.0 37.5 29.6 34.4 33.0 2.860-144 30.8 34.2 32.3 37.9 30.1 34.7 33.3 2.900-168 31.7 34.5 32.5 38.1 30.3 34.9 33.6 2.78
ND Not detectable.
SD Standard deviation.
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Antiviral
Alone
Antiviral +
GF120918 CSFBrain
CSF
Brain
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
Brain/Blood
CSF/Blood
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/Blood
CSF/Blood
Brain/Blood
CSF/Blood
Use of WBA to Assess Effect of GF120918
on Antiviral Distribution in Rats
Courtesy of Glaxo, Inc.
Toxicokinetic Modeling
Classic Compartmental Modeling
– 1-Compartment Model
– Multi-compartment Model
Physiologic Compartmental Modeling
– Perfusion-Limited Compartments
– Diffusion-Limited Compartments
– Specialized Compartments
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Disposition of Pafuramidine/Furamidine in Rat
Isolated Perfused Liver (IPL) and Sandwich-Cultured
Hepatocytes (SCH)IPL SCH
0.001
0.01
0.1
1
10
0 8 16 24M
ed
ium
(μ
M)
/ C
ell
s
(nm
ol)
Time (h)
0.001
0.01
0.1
1
10
0 8 16 24
0.001
0.01
0.1
1
10
0 8 16 24
Medium & Cells: n = 3 rats / timepoint
0.001
0.01
0.1
1
10
0 8 16 240.001
0.01
0.1
1
10
0 20 40 60 80 100 120
Furamidine (Perfusate
/ Medium)
Perfusate: n = 3-6 rats / timepoint
Liver: n=1 rat / timepoint
0.001
0.01
0.1
1
10
0 20 40 60 80 100 120
Furamidine (Liver/Cells) M3 (Perfusate/Medium) Pafuramidine (Perfusate/Medium)
Pe
rfu
sa
te (μ
M)
/ L
ive
r (μ
mo
l)
Time (min)
0.001
0.01
0.1
1
10
0 20 40 60 80 100 120
0.001
0.01
0.1
1
10
0 20 40 60 80 100 120
Initial concentration: 10 μM
99%
99%
Yan et al. J Pharmacol Exp Ther, in review, 2010
Time (min) Time (h)
Scheme Describing Disposition of Prodrug and
Metabolite in Isolated Perfused Liver and Sandwich-
Cultured Hepatocytes
Yan et al. J Pharmacol Exp Ther, in review, 2010
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Intrahepatic Binding Markedly Influences
Disposition of Active Metabolite
Yan et al. J Pharmacol Exp Ther, in review, 2010
• Hepatic accumulation of active metabolite was extensive (>95% of total formed)
• Hepatic unbound fraction (fu,L) of furamidine was only 0.3% explaining, at
least in part, low perfusate (systemic) exposure of furamidine.
Schematic Structure of Physiologic Toxico-
kinetic Model for Inhaled Propylene Oxide
Csanády G A , Filser J G
Toxicol. Sci. 2006;95:37-62