Biochemical and Molecular Toxicology

ENVR 442; TOXC 442; BIOC 442

Principles of
Toxicokinetics/Toxicodynanics

Kim L.R. Brouwer, PharmD, PhD

kbrouwer@unc.edu; 919-962-7030
 Pharmacokinetics/
Toxicokinetics:

the study of the time course of

xenobiotic absorption,
distribution, metabolism and
excretion
 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
Pharmacokinetics/ Pharmacodynamics/
Toxicokinetics Toxicodynamics

Plasma Site
Dosage Effects:
Concen- of Pharmacologic
Regimen/
tration Action Toxic
Exposure
 Toxicokinetic/ADME Studies
 Pharmacokinetics / Bioavailability
 Mass Balance
 Tissue Distribution
 Metabolite Profile
 Plasma Protein Binding
 Inhibition / Induction
Pre-Clinical
Clinical Testing
Testing

Dose/Response
Pop’n PK Bio-
PK-Guided In Large Equivalence
Dose Efficacy
In Vitro
Escalation Trials Generics
PK / PD Efficacy
Special Product line
Populations extension
Animal Dose Selection
PK / PD
Safety Post
Assessment Marketing
Patient
Variables
Toxicology Phase 3
Phase 2
Phase 1
 Terms used in
Pharmacokinetics/Toxicokinetics:
 Cmax  Vdss
 Tmax  fu
λ  Cl
 t 1/2  MRT
 AUC  MAT
 AUMC F
 Cmax
maximum concentration of compound
observed in the matrix of interest

 Tmax
time of maximum concentration
 Cmax
10000
Concentration

1000

Tmax - time of maximum concentration

100
0 5 10 15 20 25

Time
 lambda (λ)
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%
 ln 2
t1 =
10000 2 λ
Concentration

λ
1000
- slope =

100
0 5 10 15 20 25

Time
 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
0.693 xV
t1 =
2 Cl
 AUC

ln Concentration
area under the
concentration
vs time curve
AUC

Time ∞
 AUC 0→∞ = AUC tau

Single Dose Multiple Dose to Steady-State

ln Concentration

Time ∞

tau
Time
 Steady-state
 At Steady-State:
Rate of input = Rate of elimination
 Time * Concentration
 AUMC
area under the
first moment
curve
AUMC

Time ∞
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)
 Mathematically:
– volume of fluid
(usually blood) from
which compound is Dose
removed completely Cl =
per unit time
AUC∞

 Organs that may be involved in clearance:

» GI tract
» Liver
» Kidney
» Lungs
» Other sites (e.g., blood, skin)
Clearances (Cl) are Additive
Pharmacokinetic Parameters of
GI158104X in Dog

Dose ClIV Clr Clother

mg/kg/day (mL/min/kg) (mL/min/kg) (mL/min/kg)

9.5 10 0.7 9.3

 Linear Pharmacokinetics
 Linear Kinetics = Dose-Proportional kinetics
 AUC (or concentration) changes
proportionally with dose

AUC

Dose
Avandia Package Insert
 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:

Dose * AUMC
Vd ss = 2
AUC
 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 Blood
– the unbound Bound Unbound
Drug Drug
concentration is in
equilibrium between the
tissues and blood
Bound Unbound
Drug Drug

Tissue
Protein-Binding Changes May be Clinically
Relevant for the Following Types of
Compounds and Routes of Administration
High Extraction Low Extraction
Ratio 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)

Q Clearing Q
Cin Cout
Organ

Drug Elimination
Cin - Cout
E =
Cin
 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 vitro
measures 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:

AUCPO DoseIV
F= *
AUCIV DosePO
 Relative Bioavailability
fraction of the dose of a test product
that reaches the systemic circulation
relative to a reference product

 Mathematically:

AUC Dose
F= test * ref
AUC Dose
ref test
Bioavailability is an important
determinant of pharmacologic
response and therapeutic
effectiveness
Estimation of Oral Bioavailability (F)
After an IV and Oral Dose of
Compound X
Dose AUCIV 1 AUCPO 1
F
mg/kg/day (µg*hr/mL) (µg*hr/mL)

5.0 6.25 5.94 0.95

25.0 32.4 8.15 0.25

1 mean, n=4
 Factors Responsible for Poor
or Variable Oral Bioavailability

 Physicochemical Factors
 Physiologic Factors
 Influence of Formulation Factors
on the Concentration-Time Profile

Rosiglitazone Plasma Concentration, ng/mL

 Formulations with 200
E
different release rates GH
G
E F
C
D
Formulation #1
Formulation #2
H D C
E E Formulation #3

(absorption kinetics) 150 H

H G F
H

E
G
F
D
F
G
Formulation #4
Formulation #5
G H H Formulation #6
D E
C
FFF C F
HG G
100 E D
D H
FE
GE G E
C
H F
C
H D D
C E
F C
F
50 G G D
E D H
G
H
H C
GF D
EED E
F
C
HFD CC
G D
G
H
FD C
E C
E
0 H
G
FC
E
D
CD C
0 4 8 12 16 20 24 28 32 36

Time, h
 Influence of Formulation on the
Bioavailability of a Compound

Serum Concentration vs Time Profile

 Bioequivalence
250
Concentration (ng/mL)

studies are
200
performed to select
150 Form. A
Form. B
the formulation
100
which gives the
50
greatest oral
0
0 5 10 15 exposure to the drug
Time (hours)
 Special Studies:
Effects of Food on Exposure
AUC (inf)
(ng*hr/mL)

4500
 Often the fed/fasted
4000 state of the
3500 animal/human can
3000
2500
influence the
2000 absorption of drugs
1500
 Food can enhance or
1000
500 inhibit absorption
0
 Food can impact
Fed Fasted
clinical dosing
Physiologic Factors Responsible
for Poor or Variable Oral
Bioavailability
 CellMembranes
 pH, Volume and Content of GI Fluid
 GI Motility
 Vascularity and Blood Flow
 Enzymatic Degradation / Metabolism
 Disease States
The Gastrointestinal Tract
Factors Affecting Systemic Availability
Gut Gut
Portal Vein
Lumen Wall Protein Binding
RBC Partitioning
Blood/Plasma Stability
Systemic
Circulation

Solubility Liver
Chemical Stability
Permeability
Metabolism
Mucosal Metabolism
Biliary Excretion
Transport/Efflux

Metabolism

Feces
 Presystemic Elimination: (First-Pass Effect)
Loss or metabolism of drug between the administration and sampling site
Example: Grapefruit Juice Decreases Felodipine Bioavailability
 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:
Assessing Sites of Presystemic
Elimination
Dose Dose

Dose
Stomach
Portal Vein

Duodenum Systemic
Circulation

Liver
Jejunum
& Ileum
Additional Studies for Compounds
That Exhibit Poor Bioavailability
 In Vivo Portal Vein Administration Studies:

F PV = AUCPV * DoseIV
AUCIV DosePV

– 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
Assessing Sites of Presystemic
Elimination
Dose Dose

Dose Dose
Stomach
Portal Vein

Duodenum Systemic
Circulation

Liver
Jejunum
& Ileum
 Case Study: Assessing Sites of
Presystemic Elimination
Route of AUC Bioavailability
Administration (µg*hr/ml) (%)
Oral 972 21

Intraduodenal 1378 30

Portal Vein 4386 94

Intravenous 4653 100

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
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

Dose Cmax 1 AUC 1

mg/kg/day (ug/mL) (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
 Dose Selection

12000
10000 Rat
8000 Dog
AUC
6000
(ng*h/mL)
4000
2000
0
0 100 200 300 400
Dose (mg/kg/day)
Saturation of Absorption
40

Bioavailability (%)
35 Mouse Dog
30
25
20
15
10
5
0
25 500 10 500
Dose (mg/kg)
Toxicokinetics: Dose Proportionality
A = AUC increases 1600
proportional to dose. C
1400

B = Less than 1200

proportional increase in 1000

exposure: Absorption- AUC hr*µg/m 800 A

limited exposure. 600

C = Greater than 400

B
proportional increase in 200

exposure: Capacity- 0
0 50 100 150 200 250 300
limited elimination Dose (mg/kg)

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
 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 Rats
Age

13 Weeks (n=7) 26 Weeks (n=11) 52 Weeks (n=7)

Parameters
Mean CV% Mean CV% Mean CV%
Half-life
1.66 12.1 1.82 14.2 2.29 42.3
(hr)
C (0) 19.0 12.1 27.7 21.6 46.9 21.8
(ug/mL)
AUC
45.3 18.2 72.6 27.0 156.0 51.4
(ug*hr/mL)
Cl
2.01 14.8 1.29 23.5 0.664 71.4
(L/hr/kg)
Vd
4.75 11.2 3.35 22.0 1.98 24.9
(L/kg)

From: S. Garattini, Drug Metabolism Reviews 13, (3), 1982

Time (Exposure) Dependence
40
35
30
Inhibition
 Induction and Inhibition
25 Normal
20
15
Induction of metabolism as
10
5 examples of elimination
0
0 5 10 15 20 25
Time changes
100

Inhibition
Normal
 Induction or Inhibition
Induction
10
may occur without
affecting the overall
1
0 5 10
Time
15 20 25
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
 Exposure Verification
Concentration (ng/mL) 10000

1000

Males
100 Females
Expected

10

1
0 50 100 150 200
Time (h)
 Special Toxicokinetic Issues
 Toxicity / Potency Evaluations
 Metabolic Sites
 Correlation of Residual Drug with Toxicity
 Species Selection
 Gender Effects
 Placental Transfer
 Milk Transfer
 Coverage for
Human Metabolites
 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
From: Ferrero, J.L. and Brendel, K. Advances in
Pharmacology V43, pp131-169, 1997.
 Use of Whole Body Autoradiography
(WBA) to Assess Effect of GF120918 on
Antiviral Distribution in Rats
Antiviral
Brain
Alone
CSF 0.86
0.90
Ratio
0.80

0.70

0.60

0.50 0.35
Ratio
0.45
Blood 0.40

Antiviral + Brain 0.30

GF120918 CSF 0.20

0.06 CSF/Blood
0.10

0.00
Brain/Blood
Antiviral Alone
Antiviral + GF120918

Brain/Blood
CSF/Blood
 Toxicokinetic Modeling
 Classic Compartmental Modeling
– 1-Compartment Model
– Multi-compartment Model

 Physiologic Compartmental Modeling

– Perfusion-Limited Compartments
– Diffusion-Limited Compartments
– Specialized Compartments
 Scheme Describing Disposition of Prodrug and
Metabolite in Isolated Perfused Liver and Sandwich-
Cultured Hepatocytes

Yan et al. J Pharmacol Exp Ther, 337:503-512, 2011

 Intrahepatic Binding Markedly Influences
Disposition of Active Metabolite

• 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.
Yan et al. J Pharmacol Exp Ther, 337:503-512, 2011
 Schematic of Physiologically-Based
Pharmacokinetic Model

1. Parameters
» Molecular Weight
» Log P
» Partition Coefficients
» Protein Binding
» Metabolic (Vmax & Km)
» Clearance
2. Structure
– Organs & tissues involved
– Organ Weights
– Organ Flows
– Perfusion/Diffusion and/or
Transport
3. Equations
– Mass Balance
– Clearance from tissues
 Schematic Structure of Physiologically-Based
Toxicokinetic Model for Inhaled Propylene Oxide

Csanády G A , Filser J G
Toxicol. Sci. 2006;95:37-62