Battelle
Draft
June,
2002
7
PRE­
VALIDATION
STUDY
PLAN
AND
STUDY
PROTOCOL
FOR
THE
AROMATASE
ASSAY
USING
HUMAN,
BOVINE,
AND
PORCINE
PLACENTA,
AND
HUMAN
RECOMBINANT
MICROSOMES
EPA
Contract
Number
68­
W­
01­
023
WA
2­
5,
Task
12
June
10,
2002
PREPARED
FOR
GARY
E.
TIMM
WORK
ASSIGNMENT
MANAGER
U.
S.
ENVIRONMENTAL
PROTECTION
AGENCY
ENDOCRINE
DISRUPTOR
SCREENING
PROGRAM
WASHINGTON,
D.
C.

BATTELLE
505
KING
AVENUE
COLUMBUS,
OH
43201
Battelle
Draft
i
June,
2002
TABLE
OF
CONTENTS
Page
INTRODUCTION
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1
1.0
ENDPOINT
MEASUREMENTS
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2
1.1
AROMATASE
REACTION
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2
1.2
HUMAN
PLACENTAL
MICROSOMAL
AROMATASE
ASSAY
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3
1.3
OTHER
MICROSOMAL
AROMATASE
ASSAYS
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3
1.4
METHODS
FOR
MEASURING
AROMATASE
ACTIVITY
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4
2.0
PROTOCOL
RESOLUTION
ISSUES/
STUDY
DESIGN
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5
2.1
PRE­
OPTIMIZATION
EXPERIMENTS
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5
2.2
AROMATASE
ASSAY
OPTIMIZATION
EXPERIMENTS
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5
3.0
OPTIMIZED
ASSAY
EXPERIMENTS
USING
SELECTED
TEST
SUBSTANCES
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6
4.0
STUDY
PROTOCOL
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8
5.0
STATISTICAL
METHODS
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8
5.1
OPTIMIZATION
EXPERIMENTS
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8
5.1.1
Experimental
Factors
and
Response
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8
5.1.2
Design
Considerations
for
the
Microsomal
Preparations
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9
5.1.3
Experimental
Design
Matrix
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9
5.1.4
Statistical
Analysis
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10
5.1.5
Determine
Variability
of
the
Optimized
Assays
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11
5.2
OPTIMIZED
ASSAY
USING
SELECTED
TEST
SUBSTANCES
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11
5.2.1
Test
Run
Composition
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11
5.2.2
Quality
Control
Comparisons
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12
5.2.3
Concentration
Response
Curve
Fits
to
the
Standard
Tests
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12
5.2.4
Concentration
Response
Curve
Fits
to
the
Test
Substances
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13
5.2.5
Comparison
of
Optimized
Assays
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14
5.2.6
Comparisons
Based
on
Reduced
Numbers
of
Test
Concentrations
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14
6.0
REFERENCES
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15
APPENDIX
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A­
1
Battelle
Draft
1
June,
2002
Endocrine
Disruptor
Screening
Program
Contract
No.
68­
W­
01
 
023
Work
Assignment
2­
5,
Task
12
Aromatase
Pre­
validation
Study
Plan
INTRODUCTION
The
objective
of
the
aromatase
assay
is
to
detect
the
ability
of
environmental
chemicals
to
inhibit
the
enzyme
aromatase.
Estradiol
is
biosynthesized
from
androgens
by
the
cytochrome
P450
enzyme
complex
called
aromatase
and
the
levels
of
estrogens
are
controlled
by
the
extent
of
formation
of
estrogens
from
androgens.
The
levels
of
estradiol,
the
most
potent
endogenous
estrogen
in
humans,
are
critical
for
female
reproduction
and
other
hormonal
effects
in
females.
Alterations
in
the
amount
of
aromatase
present
or
in
the
catalytic
activity
of
the
enzyme
will
alter
the
levels
of
estrogens
in
tissues
and
dramatically
disrupt
estrogen
hormone
action.
Inhibition
of
aromatase
alters
the
catalytic
activity
of
the
enzyme
and
results
in
a
rapid
decrease
in
the
levels
of
estrogens.

An
aromatase
assay
is
proposed
as
one
of
the
Tier
1
Screening
Battery
Alternate
Methods.
If
the
20­
day
pubertal
female
assay
is
not
selected
as
a
primary
Tier
1
Screening
Method,
the
20­
day
male
pubertal
assay
will
be
used
as
an
alternative.
The
aromatase
assay
would
be
used
to
complement
the
male
pubertal
assay.
An
in
vitro
aromatase
assay
could
easily
be
utilized
to
assess
the
effects
of
various
environmental
toxicants
on
aromatase
activity.
Environmental
chemicals
and
various
natural
products
can
inhibit
aromatase
activity
through
a
direct
alteration
of
the
catalytic
activity
of
the
enzyme,
resulting
in
a
rapid
decrease
in
the
levels
of
estrogens.

The
pre­
validation
study
plan
addresses
the
optimization
of
the
aromatase
assay
using
human
placental
microsomal
preparations,
bovine
placental
microsomal
preparations,
porcine
placental
microsomal
preparations,
and
recombinant
human
aromatase
microsomal
preparations.
This
pre­
validation
study
plan
has
been
developed
to
address
the
following
issues:

°
the
endpoints
to
be
measured
°
the
protocol
issues
needing
resolution
°
the
study
design
to
address
the
protocol
issues
°
recommended
test
substances
and
concentrations
to
be
used
and
the
justification
for
each
recommendation
°
the
detailed
study
protocols
°
statistical
methods
for
comparing
the
performance
of
the
assays.
Battelle
Draft
2
June,
2002
HO
O
O
O
androstenedione
O
O
estrone
Reaction
Mechanism
for
Estrogen
Biosynthesis
by
Aromatase
HO
O2
NADPH
O
O
HO
O2
NADPH
HO
­
H2O
O
O
O
19­
hydroxyandrostenedione
19,19­
dihydroxyandrostenedione
19­
oxoandrostenedione
O2
NADPH
O
O
HO
O
O
+
3Fe
­
H2O
­
HCOOH
peroxy
enzyme
intermediate
1.0
ENDPOINT
MEASUREMENTS
1.1
AROMATASE
REACTION
Aromatase
is
the
enzyme
complex
responsible
for
the
conversion
of
androgens
to
estrogens
during
steroidogenesis
(
Simpson
et
al.,
1994).
The
enzyme
complex
is
bound
in
the
endoplasmic
reticulum
of
the
cell
and
is
comprised
of
two
major
proteins
(
Simpson
et
al.,
Kellis
and
Vickery,
1987).
One
protein
is
cytochrome
P450
arom,
a
hemoprotein
that
converts
C
19
steroids
(
androgens)
into
C
18
steroids
(
estrogens)
containing
a
phenolic
A
ring.
The
second
protein
is
NADPH­
cytochrome
P450
reductase,
which
transfers
reducing
equivalents
to
cytochrome
P450
arom.
Three
moles
of
NADPH
and
three
moles
of
oxygen
are
utilized
in
the
conversion
of
one
mole
of
substrate
into
one
mole
of
estrogen
product
(
reaction
is
illustrated
below).
Aromatization
of
androstenedione,
the
preferred
substrate,
proceeds
via
three
successive
oxidation
steps,
with
the
first
two
being
hydroxylations
of
the
angular
C­
19
methyl
group.
The
final
oxidation
step,
whose
mechanism
remains
for
complete
elucidation,
proceeds
with
the
aromatization
of
the
A
ring
and
loss
of
the
C­
19
carbon
atom
as
formic
acid.
Battelle
Draft
3
June,
2002
1.2
HUMAN
PLACENTAL
MICROSOMAL
AROMATASE
ASSAY
The
human
placental
microsomal
assay
is
commonly
used
for
measuring
the
inhibition
of
aromatase
activity
by
various
compounds,
including
potential
therapeutic
agents
and
various
endocrine
disruptors
(
Lephart
and
Simpson,
1991).
The
source
of
the
aromatase
is
a
microsomal
preparation
isolated
from
human
term
placenta.
Normal
placenta
is
obtained
after
delivery
at
a
local
hospital,
is
delivered
on
ice
to
the
laboratory,
and
then
processed
at
4

C
following
a
standard
procedure.
The
microsomal
preparation
is
isolated
by
differential
centrifugation
procedures.
This
microsomal
preparation
consists
of
the
endoplasmic
reticulum
membrane
of
the
cell
and
contains
the
membrane­
bound
cytochrome
P450
arom
and
the
NADPH­
cytochrome
P450
reductase.
Complete
enzyme
activity
requires
the
addition
of
either
NADPH
or
an
NADPHgenerating
system,
and
the
activity
is
measured
using
either
the
product
isolation
method
or
the
radiometric
method.
The
highest
tissue
levels
of
human
aromatase
are
present
in
term
placenta
and
the
placenta
is
discarded
after
birth,
thus
providing
a
rich
and
inexpensive
source
of
the
enzyme.
Consequently,
the
human
placental
microsomal
aromatase
assay
is
used
extensively
in
academic
labs
and
pharmaceutical
firms
as
the
initial
biological
evaluation
for
potential
steroidal
and
nonsteroidal
aromatase
inhibitors.

1.3
OTHER
MICROSOMAL
AROMATASE
ASSAYS
Aromatase
has
also
been
well
studied
in
other
mammalian
species
(
rodents,
cows,
pigs,
horses)
and
in
non­
mammalian
vertebrates.
In
many
species,
aromatase
expression
and/
or
activity
is
restricted
to
the
gonads
and
the
brain.
Placental
aromatase
is
also
found
in
other
primates,
cattle,
horses,
sheep,
and
pigs.
Microsomal
preparations
from
placental
tissues
from
these
animals
are
used
in
investigations
of
comparative
biochemical
endocrinology,
gene
expression
studies,
and
structure­
function
studies
of
cytochrome
P450
arom.
Similar
to
humans
and
other
primates,
cattle
and
sheep
have
a
single
functional
CYP19
gene
and
the
gene
is
regulated
by
tissue­
specific
promoter
regions
(
Vanselow,
et
al.,
2001).
Pigs,
on
the
other
hand,
have
several
aromatase
proteins
that
are
produced
by
three
distinct
genes
(
Conley
et
al.,
1996;
Corbin
et
al.,
1995;
Corbin
et
al.,
1999).

Biochemical
studies
have
shown
species
differences
in
regards
to
substrate
specificity
and/
or
inhibition
by
various
steroidal
and
nonsteroidal
agents,
suggesting
catalytic
differences
at
the
enzyme
active
site
as
a
result
of
amino
acid
sequence
differences.
In
pigs,
testosterone
is
aromatized
more
efficiently
(
apparent
K
m
for
testosterone
is
33
nM,
apparent
K
m
for
androstenedione
is
77
nM)
and
differences
in
sensitivity
to
imidazole
inhibitors
have
been
reported
(
Corbin
et
al.,
1995;
Conley
et
al.,
1997).
Differences
in
inhibition
by
aniline
inhibitor
analogs
have
been
observed
in
bovine
vs.
human
placental
enzymes
(
Kellis
and
Vickery,
1985).
Similarly,
imidazole
and
indolizinone
derivatives
have
different
aromatase
inhibition
kinetics
in
equine
vs.
human
placental
preparations
(
Auvray
et
al.,
1999;
Moslemi
and
Seralini,
1997;
Moslemi
et
al.,
1998).

Recombinant
systems
for
expression
of
aromatase
have
also
been
developed
using
a
cDNA
clone
of
human
placental
aromatase
containing
a
baculovirus
expression
system
in
insect
Battelle
Draft
4
June,
2002
cell
suspension
cultures
(
Lahde
et
al.,
1993;
Sigle
et
al.,
1994;
Amarneh
and
Simpson,
1995).
In
general,
these
systems
produce
catalytically
active
aromatase
enzyme,
and
the
levels
of
enzyme
activity
in
insect
microsomal
preparations
are
similar
to
the
levels
in
human
placental
microsomal
preparations.
Such
systems
are
primarily
used
to
generate
sufficient
aromatase
protein
for
enzyme
purification.
A
commercial
microsomal
preparation
of
recombinant
human
aromatase
from
stably­
transfected
insect
cells
is
available
and
has
been
used
in
a
potential
high­
throughput
assay
(
McNamara
et
al.,
1999).

1.4
METHODS
FOR
MEASURING
AROMATASE
ACTIVITY
The
method
employed
for
measuring
aromatase
activity
in
vitro
and
in
vivo
is
critical.
This
assay
endpoint,
i.
e.,
measurement
of
aromatase
activity,
must
be
accurate
and
reproducible.
The
endpoint
is
accurate
when
the
assay
measurement
is
in
agreement
with
the
accepted
reference
value.
The
endpoint
is
reproducible
when
the
same
findings
occur
under
the
same
conditions
within
a
single
laboratory
(
intra­
laboratory)
and
among
other
laboratories
(
inter­
laboratory).
The
two
methods
that
are
utilized
extensively
for
the
determination
of
aromatase
activity
in
both
in
vitro
and
in
vivo
assays
are
the
product
isolation
method
and
the
radiometric
method
(
Lephart
and
Simpson,
1991).

The
most
rigorous
method
is
the
product
isolation
technique.
The
method
involves
administration
of
a
substrate
such
as
androstenedione
or
testosterone,
incubation
or
treatment
for
a
designated
time,
isolation
of
the
estrogen
products
formed,
and
measurement
of
the
amount
of
estrogens.
One
common
method
uses
radiolabeled
substrate
(
either
3H
or
14C)
in
the
aqueous
incubation
medium.
At
the
end
of
the
incubation
or
treatment
period,
isolation
of
the
radiolabeled
steroids
is
accomplished
by
organic
solvent
extraction
techniques.
The
radiolabeled
substrate
and
products
are
separated
using
either
thin
layer
chromatography
or
high­
pressure
liquid
chromatography
(
HPLC)
and
analysis
of
the
quantity
of
estrogen
products
formed
using
liquid
scintillation
counting.
This
method
is
best
suited
for
in
vitro
assays
using
subcellular
enzyme
preparations,
tissue
incubations,
or
cell
culture
systems.
Variations
of
the
product
isolation
assay
method
include
unlabeled
substrate
(
or
endogenous
substrate
in
an
in
vivo
study)
and
other
methods
for
measurement
and
quantification.
These
other
measurement
methods
include
mass
spectrometry,
radioimmunoassays
(
RIA),
or
enzyme­
linked
immunoassays
(
EIA).

The
radiometric
method
is
also
utilized
for
in
vitro
measurements
of
aromatase
activity.
This
method
is
also
referred
to
as
the
3H
2
O
assay.
The
level
of
aromatase
activity
is
determined
by
measuring
the
amount
of
3H
2
O
released
from
[
1b­
3H]­
androstenedione
substrate.
The
basis
of
this
assay
is
that
the
aromatization
of
the
A­
ring
catalyzed
by
aromatase
involves
the
stereospecific
cleavage
of
the
covalent
bond
between
the
carbon
atom
at
position
1
and
the
hydrogen
on
the
b
face
of
the
steroid
ring
system.
The
procedures
for
this
method
are
similar
to
the
product
isolation
method
but
do
not
involve
any
chromatography
to
separate
steroid
substrate
and
products.
Rather,
the
amount
of
3H
2
O
released
is
measured
in
the
aqueous
phase
following
rigorous
extractions
with
organic
solvents
and/
or
dextran­
coated
charcoal.
This
method
is
very
straightforward
and
more
rapid.
However,
the
results
of
the
radiometric
method
must
be
confirmed
for
the
specific
aromatase
assay
conditions
using
the
product
isolation
assay
before
Battelle
Draft
5
June,
2002
extensive
use.
This
radiometric
method
has
been
confirmed
for
in
vitro
assays
using
human
placental
microsomes,
ovarian
microsomes
and
tissues,
cell
culture
systems
(
including
human
breast
cancer
cells
and
human
choriocarcinoma
cells),
and
isolated
human
breast
or
ovarian
cells.
This
comparison
of
the
results
from
the
radiometric
method
with
the
product
isolation
assay
is
critical
because
other
biochemical
pathways
that
involve
extensive
androgen
metabolism
without
aromatization
of
the
androgen
can
produce
false
positive
measurements.
Examples
are
determinations
of
3H
2
O
released
by
liver
or
prostate
cells,
tissues,
or
tissue
homogenates,
which
results
in
much
higher
activity
measurements
than
is
observed
with
a
product
isolation
method
as
a
result
of
androgen
metabolism,
and
presence
of
a
19­
hydroxylase
activity
in
adrenal
tissues
which
does
not
result
in
A
ring
aromatization
and
formation
of
estrogens.

2.0
PROTOCOL
RESOLUTION
ISSUES/
STUDY
DESIGN
2.1
PRE­
OPTIMIZATION
EXPERIMENTS
The
pre­
optimization
experiments
are
designed
to
assess
the
chemical
and
biological
properties
of
the
critical
components
that
are
used
in
the
aromatase
assay.
These
experiments
include
characterizing
the
radiolabeled
substrate
and
preparation
of
the
placental
microsomes.
In
addition,
the
microsomal
preparations,
including
the
human
recombinant
microsomes,
will
be
analyzed
for
protein
concentration,
cytochrome
P450
content,
and
aromatase
activity.
The
P450
content
measurement
will
provide
assurance
that
the
enzyme
is
present
(
and
in
what
concentration/
preparation
type)
prior
to
beginning
the
more
elaborate
aromatase
activity
assay.
A
single
aromatase
activity
assay
using
each
type
of
microsomal
preparation
was
included
as
a
preoptimization
experiment
in
order
to
determine
whether
the
preparations
are
of
sufficient
activity
to
conduct
the
optimization
experiments.
The
outcome
of
the
pre­
optimization
experiments
will
be
to
know
that
the
placental
and
recombinant
microsomes
and
substrate
are
sufficient
in
quantity
and
activity
to
conduct
the
optimization
experiments.

2.2
AROMATASE
ASSAY
OPTIMIZATION
EXPERIMENTS
The
assay
optimization
experiments
are
designed
to
optimize
the
chemical
and
biological
components
of
the
microsomal
aromatase
assay.
These
experiments
involve
a
factorial
design
approach
to
include
evaluation
of
co­
factor
concentration,
enzyme
concentration,
protein
concentration,
substrate
concentration,
and
incubation
time
using
human
placental
microsomal
preparations,
bovine
placental
microsomal
preparations,
porcine
placental
microsomal
preparations,
and
recombinant
human
aromatase
microsomal
preparations.
This
experiment
will
include
four
parts.

°
Part
1,
26­
1
factional
runs,
will
permit
the
estimation
of
all
linear
main
effects
and
linear
by
linear
interactions
to
be
determined.
Battelle
Draft
6
June,
2002
°
Part
2,
center
point
run,
will
provide
an
estimate
of
the
response
in
the
center
of
the
design
space
and
an
overall
indication
of
the
goodness­
of­
fit
to
the
linear
trend
assumptions.

°
Part
3,
axial
point
runs,
will
provide
an
estimate
of
the
quadratic
main
effects
for
each
of
the
factors.

°
Part
4,
replicate
runs,
will
provide
an
estimate
of
the
reproducibility
of
the
response
at
the
center
of
the
design
space
and
at
various
extremes
of
the
design
space.

In
addition,
this
stage
of
the
study
plan
will
also
provide
an
estimate
of
the
variability
of
the
optimized
assay
in
each
of
the
different
microsomal
preparations.
Thus,
the
outcome
of
the
optimization
experiments
will
be
to
know
the
optimal
conditions
and
variability
of
the
aromatase
assay
in
each
of
the
microsomal
preparations
in
order
to
test
the
assay
using
selected
test
substances.

3.0
OPTIMIZED
ASSAY
EXPERIMENTS
USING
SELECTED
TEST
SUBSTANCES
Various
substances
will
be
tested
in
the
optimized
aromatase
assays
using
human
placental
microsomal
preparations,
bovine
placental
microsomal
preparations,
porcine
placental
microsomal
preparations,
and
recombinant
human
aromatase
microsomal
preparations.
The
results
of
these
evaluations
will
be
compared
to
determine
whether
the
assays
were
similar
or
not
in
their
response
to
detect
an
effect
of
the
test
substance.

For
each
of
the
four
optimized
assays,
seven
test
substances
that
exhibit
aromatase
inhibition
to
varying
degrees
will
be
evaluated:

°
Aminoglutethimide:
This
compound
is
a
non­
steroidal
aromatase
inhibitor
exhibiting
moderate
inhibitory
activity.
It
is
referred
to
as
a
first­
generation
aromatase
inhibitor
and
was
the
first
agent
used
for
inhibition
of
aromatase
in
clinical
trials.
Aminoglutethimide
is
commonly
used
in
various
in
vitro
assays
as
a
standard
inhibitor
to
compare
results
with
other
non­
steroidal
agents.
IC
50
values
for
aromatase
inhibition
by
aminoglutethimide
in
in
vitro
assays
typically
range
from
1.0
to
6.0
µ
M.
This
agent
is
commercially
available.

°
Letrozole
or
Anastrozole:
These
compounds
are
non­
steroidal
aromatase
inhibitors
exhibiting
potent
inhibitory
activity.
Letrozole
is
marketed
by
Novartis
under
the
trade
name
Femara,
and
anastrozole
is
marketed
by
Astra
Zeneca
under
the
trade
name
Arimidex.
These
agents
are
referred
to
as
third­
generation
aromatase
inhibitors.
Both
drugs
are
approved
as
first­
line
therapy
in
women
with
advanced
hormone­
dependent
breast
cancer
and
as
second­
line
therapy
in
women
with
hormone­
dependent
breast
cancer
who
have
failed
tamoxifen
treatment.
IC
50
values
for
aromatase
inhibition
by
these
two
agents
in
in
vitro
assays
have
been
reported
in
the
low
nanomolar
range
(
1
 
Battelle
Draft
7
June,
2002
15
nM),
and
both
agents
suppress
estrogen
biosynthesis
in
vivo
by
greater
than
96%.
The
only
sources
for
these
agents
are
the
pharmaceutical
manufacturers,
and
it
may
be
difficult
to
obtain
these
agents
for
that
reason.

°
4­
Hydroxyandrostenedione:
This
compound
is
a
steroidal
aromatase
inhibitor
exhibiting
potent
inhibitory
activity.
It
is
referred
to
as
a
second­
generation
aromatase
inhibitor
and
was
the
second
agent
used
for
inhibition
of
aromatase
in
clinical
trials.
4­
Hydroxyandro­
stenedione
(
4­
OH­
A,
formestane)
is
commonly
used
in
various
in
vitro
assays
as
a
standard
inhibitor
to
compare
results
with
other
steroidal
agents.
IC
50
values
for
aromatase
inhibition
by
4­
OH­
A
in
in
vitro
assays
typically
range
from
30.0
to
50.0
nM.
This
agent
is
commercially
available.

°
Chrysin:
This
compound
is
a
flavonoid
natural
product
isolated
from
various
plant
sources.
It
exhibits
moderate
aromatase
inhibitory
activity
in
in
vitro
assays,
with
IC
50
values
for
aromatase
inhibition
typically
ranging
from
0.5
to
10.0
µ
M.
This
agent
is
commercially
available.

°
Genistein:
This
compound
is
an
isoflavonoid
natural
product
isolated
from
various
plant
sources.
It
exhibits
weak
aromatase
inhibitory
activity
in
in
vitro
assays,
with
IC
50
values
for
aromatase
inhibition
typically
ranging
from
30.0
to
100.0
µ
M.
Genistein
at
micromolar
concentrations
exhibits
many
other
biological
activities
as
well,
including
weak
estrogenic
activity,
binds
to
estrogen
receptor
a
and
estrogen
receptor
b,
and
inhibits
protein
tyrosine
kinase
activity.
This
agent
is
commercially
available.

°
Econazole:
This
compound
is
an
imidazole
anti­
fungal
agent
and
is
marketed
by
numerous
pharmaceutical
or
agricultural
firms.
It
exhibits
potent
aromatase
inhibitory
activity
in
in
vitro
assays,
with
IC
50
values
for
aromatase
inhibition
typically
ranging
from
30.0
to
50.0
nM.
This
agent
is
commercially
available.

°
Ketoconazole:
This
compound
is
also
an
imidazole
anti­
fungal
agent
and
is
marketed
by
numerous
pharmaceutical
or
agricultural
firms.
It
exhibits
weak
aromatase
inhibitory
activity
in
in
vitro
assays,
with
IC
50
values
for
aromatase
inhibition
greater
than
65.0
µ
M.
This
agent
is
commercially
available.

For
each
of
the
four
optimized
assays,
five
test
substances
that
do
not
exhibit
aromatase
inhibition
will
be
evaluated:

°
Atrazine:
This
compound
is
a
2­
chloro­
s­
triazine
herbicide.
Atrazine
does
not
inhibit
aromatase
enzymatic
activity.
Atrazine
and
related
2­
chloro­
s­
triazine
herbicides
have
been
reported
to
affect
aromatase
gene
expression
and
result
in
induction
of
the
enzyme
in
cell
cultures
administered
the
agent.
This
agent
is
commercially
available.
Battelle
Draft
8
June,
2002
°
Mono­(
2­
ethylhexyl)
phthalate:
This
agent
(
MEHP)
is
the
biologically
active
metabolite
of
di­(
2­
ethylhexyl)
phthalate
(
DEHP).
Phthalates
are
plasticizers
and
are
added
to
polyvinyl
chloride­
based
products
to
increase
flexibility
of
the
materials.
MEHP
does
not
inhibit
aromatase
enzymatic
activity.
However,
MEHP
has
been
reported
to
affect
aromatase
gene
expression
and
results
in
suppression
of
the
enzyme
in
cultured
granulose
cells.
This
agent
is
commercially
available.

°
Nonylphenol:
Nonylphenol
is
a
degradation
product
of
alkylphenol
polyethoxylate
detergents,
and
nonylphenol
exhibits
weak
estrogenic
activity
through
interactions
with
the
estrogen
receptor.
Nonylphenol
does
not
inhibit
aromatase
enzymatic
activity.
This
agent
is
commercially
available.

°
Lindane:
This
agent
is
an
organochlorine
insecticide,
and
lindane
and
its
isomers
affect
the
functions
of
steroid
acute
regulatory
protein
(
StAR)
and
alter
cholesterol
metabolism.
Lindane
does
not
inhibit
aromatase
enzymatic
activity.
This
agent
is
commercially
available.

°
TCDD:
TCDD,
a
dioxin,
is
a
highly
toxic
contaminant
produced
as
a
by­
product
during
the
manufacture
of
chlorinated
phenols
and
phenoxyherbicides.
One
of
TCDD's
effects
are
to
alter
the
expression
of
other
cytochrome
P450
genes
involved
in
drug
metabolism
(
such
as
CYP1A1).
TCDD
does
not
inhibit
aromatase
enzymatic
activity.
This
agent
is
commercially
available,
but
its
use
is
restricted
due
to
its
highly
toxic
effects.

4.0
STUDY
PROTOCOL
The
study
protocol
is
included
in
this
study
plan
as
an
attachment
(
See
Appendix).

5.0
STATISTICAL
METHODS
5.1
OPTIMIZATION
EXPERIMENTS
5.1.1
Experimental
Factors
and
Response
The
object
of
the
experiment
is
to
identify
the
combination(
s)
of
experimental
conditions
that
will
maximize
the
rate
of
the
aromatase
reaction.
The
combination
of
experimental
factors
that
maximizes
this
response
will
be
experimentally
determined.
The
experimental
factors
to
be
studied
and
their
ranges
of
variation
are
as
follows:
Battelle
Draft
9
June,
2002
Factor
Levels
(
Conc.)

Factor
Identificationa
Units
Factor
Designation
1
2
3
4
5
NADP
mM
X1
0.1
0.5
1
2
4
G­
6­
P
mM
X2
0.1
1
2
3
4
G­
6­
P
dehydrogenase
units
X3
0.1
0.5
1
2
4
Androstenedione
(
substrate)
nM
X4
10
25
50
100
500
Protein
mg/
mL
X5
0.01
0.05
0.1
0.5
1
Incubation
time
min
X6
10
20
30
60
120
a
Glucose­
6­
phosphate
(
G­
6­
P)

It
is
assumed
that
all
combinations
of
these
factors
are
a
priori
possible.
Since
each
of
the
factors
range
across
one
or
more
orders
of
magnitude,
consideration
will
be
given
to
expressing
response
relations
in
terms
of
transformations
of
the
factors,
such
as
logarithmic
transformations.
All
factors
are
assumed
to
be
a
priori
equally
important
so
the
suggested
design
is
symmetric
in
each
of
the
factors.
This
assumption
may
be
modified
after
analyzing
the
results
of
the
initial
experiments.

5.1.2
Design
Considerations
for
the
Microsomal
Preparations
The
microsomal
preparations
that
will
be
used
in
the
pre­
validation
plan
are:

°
Human
placenta
°
Bovine
placenta
°
Porcine
placenta
°
Human
recombinant
microsomes.

Separate
experiments
will
be
conducted
to
optimize
the
aromatase
assay
for
each
of
the
microsomal
preparations.
Initially
a
51
run
experiment
will
be
conducted
to
optimize
the
human
placenta
Aromatase
assay.
Based
on
the
results
of
the
experiments
using
the
human
placental
microsomes,
the
optimization
experiments
using
the
bovine
and
porcine
placental
microsomes,
as
well
as
the
human
recombinant
microsomes,
may
require
further
experiments
to
estimate
more
complex
structure
(
e.
g.
higher
order
trends
of
interactions)
than
initially
assumed.
Alternatively,
the
response
structure
of
the
assay
using
the
human
placental
microsomes
may
be
simpler
than
initially
assumed.
In
this
instance,
the
designs
for
the
other
microsomal
preparations
could
then
be
reduced,
thereby
decreasing
the
level
of
effort
and
time
required
to
complete
the
optimization
experiments
for
all
four
microsomal
preparations.

5.1.3
Experimental
Design
Matrix
The
suggested
experimental
design
matrix
is
based
on
a
factorial
design
that
simultaneously
varies
each
of
the
six
experimental
factors.
The
design
permits
the
estimation
of
Battelle
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10
June,
2002
linear
and
quadratic
main
effects
for
each
of
the
factors
(
averaged
across
all
the
other
factors),
as
well
as
linear
by
linear
two
factor
interactions
among
all
pairs
of
factors.
It
is
initially
assumed
that
these
trends
and
interactions
describe
the
principal
structure
in
the
response
trends.
This
assumption
will
be
re­
evaluated
after
the
initial
test
results
are
determined.
The
initial
design
will
be
supplemented
if
appropriate.

Factorial
designs
are
preferable
to
one­
factor­
at
a
time
experiments
because
they
utilize
all
the
data
to
estimate
each
of
the
effects,
rather
than
just
the
portion
of
the
data
pertaining
to
a
single
factor.
They
also
permit
estimation
of
interaction
effects,
which
indicate
if
the
response
to
varying
one
factor
depends
on
the
levels
of
one
or
more
other
factors.

It
is
assumed
that
each
of
the
factors
can
be
varied
with
equal
ease.
Thus
the
experimental
runs
will
be
completely
randomized
across
the
51
runs.
The
experimental
matrix
is
divided
into
four
parts
but
the
runs
in
these
parts
will
be
intermixed
when
conducted
in
the
laboratory.

°
Part
1
­
26­
1
factional
factorial
design
(
32
runs).
These
runs
will
estimate
the
linear
main
effects
and
linear
by
linear
interactions.
°
Part
2.
Center
Point
design
(
1
run).
This
run
will
estimate
the
response
in
the
center
of
the
design
space
and
will
provide
an
overall
indication
of
goodness­
of­
fit
to
the
linear
trend
assumptions.
°
Part
3.
Axial
Point
design
(
12
runs).
These
runs
will
estimate
the
quadratic
main
effects
for
each
of
the
factors.
°
Part
4.
Replicate
design
(
6
runs).
These
runs
will
estimate
the
reproducibility
of
the
response
at
the
center
of
the
design
space
and
at
various
extremes
of
the
design
space.

5.1.4
Statistical
Analysis
Statistical
analysis
will
be
based
on
multiple
regression
analysis.
Preliminary
graphical
displays
will
be
used
to
identify
the
nature
of
the
trends,
the
nature
of
the
response
variability,
and
the
need
for
transformations
of
the
response
or
of
the
primary
experimental
variables
X
1,
...,
X
6
full
quadratic
response
surface
model
will
be
fitted
to
the
data.
Residuals
from
the
model
will
be
examined
graphically
and
numerically
to
identify
outlying
observations,
heterogeneity
of
variability,
and
departures
from
model
assumptions.

If
several
individual
factors
or
combinations
of
factors
exhibit
particularly
strong
influence
on
the
response
outcome,
then
consideration
will
be
given
to
augmenting
the
design
with
supplemental
runs
to
study
the
trends
or
interactions
in
these
directions
in
greater
detail.
If
the
trends
or
interactions
associated
with
several
of
the
experimental
factors
are
small
or
not
significant,
then
consideration
will
be
given
to
omitting
them
from
subsequent
experiments
with
the
other
microsomal
preparations.

The
final
response
surface
model
will
be
optimized
to
determine
the
experimental
conditions
associated
with
the
optimum
response.
A
confidence
region
will
be
constructed
around
the
optimum
by
considering
the
set
of
experimental
factors
whose
associated
responses
do
Battelle
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11
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2002
not
differ
significantly
from
that
at
the
maximum.
The
optimum
condition
may
occur
at
the
interior
of
the
design
space
or
at
a
boundary.
In
the
former
case,
the
optimum
and
an
associated
confidence
region
will
be
reported.
In
the
latter
case,
it
may
be
possible
to
further
improve
the
efficiency
of
the
reaction
by
extrapolating
outside
the
design
space.
Consideration
will
be
given
to
extending
the
experimental
region
in
the
direction
of
the
(
increasing)
response
gradient
to
determine
the
extent
of
possible
improvement
in
efficiency.

Sensitivity
analysis
will
be
conducted
in
the
region
of
the
optimum
to
assess
the
effects
of
perturbations
in
experimental
factors
from
their
optimum
values
on
the
reaction
efficiency.
The
individual
factors
X
1,
...,
X
6
will
be
varied
by
differing
amounts,
e.
g.
±
5%,
±
10%,
±
20%
to
determine
the
influence
on
reaction
rate.
The
linear
combination
of
experimental
factors
corresponding
to
the
eigenvector
associated
with
the
minimum
(
i.
e.
the
most
negative)
eigenvalue
of
the
matrix
of
normalized
second
partial
derivatives
at
the
maximum
will
also
be
studied
to
assess
the
effect
on
reaction
sensitivity
of
perturbations
in
experimental
conditions
in
the
direction
of
greater
rate
of
change
of
the
response
surface.

5.1.5
Determine
Variability
of
the
Optimized
Assays
After
optimum
conditions
have
been
determined
for
the
assay
using
the
four
different
microsomal
preparations,
each
assay
will
be
rerun
at
its
optimum
conditions
to
assess
variability
of
results.
Each
assay
will
be
conducted
independently
by
three
technicians
and
at
three
separated
times
per
technician.
This
permits
the
estimation
of
the
technician­
to­
technician
and
the
within
technician
components
of
variation.
These
components
of
variation
can
be
compared
with
that
determined
from
the
regression
fit
to
assess
their
statistical
and
biological
significance.

The
variance
components
will
be
compared
across
the
assays
to
determine
their
comparability.

5.2
OPTIMIZED
ASSAY
USING
SELECTED
TEST
SUBSTANCES
5.2.1
Test
Run
Composition
This
testing
stage
of
the
study
will
be
carried
out
in
a
single
laboratory,
under
homogeneous
conditions.
The
term
"
run"
will
be
used
to
describe
when
the
assay
is
used
to
measure
aromatase
activity
of
a
single
sample.
Multiple
runs
will
be
conducted
each
day
and
the
multiple
runs
will
be
referred
to
as
a
"
batch."
The
size
of
a
batch
is
up
to
48
runs
and
the
order
of
the
runs
will
be
randomized
within
a
batch.

On
a
given
day,
the
batch
of
runs
will
include:

°
Four
replicates
(
4
runs)
of
the
substrate
standard
(
androstenedione)
at
the
highest
tested
concentration.
This
sample
will
provide
the
lowest
measure
of
aromatization
due
to
greater
substrate­
enzyme
catalysis
of
the
non­
radiolabeled
substrate
relative
to
the
radiolabeled
substrate.
It
is
designated
at
the
NSB
(
non­
specific
binding)
sample.
Battelle
Draft
12
June,
2002
°
One
replicate
of
the
substrate
standard
at
each
of
the
seven
other
concentrations
(
7
runs)

°
Four
replicates
(
4
runs)
of
the
vehicle.
The
test
substances
that
are
run
within
the
same
batch
will
be
grouped
so
that
they
use
the
same
vehicle.

The
vehicle
replicates
will
provide
the
highest
measure
of
aromatization
(
assuming
the
vehicle
is
inert)
since
it
will
not
reduce
radiolabeled
substrate­
enzyme
catalysis.

°
One
replicate
of
each
test
substance
(
up
to
four
a
day)
at
each
of
the
eight
concentrations
tested
(
up
to
32
runs).

5.2.2
Quality
Control
Comparisons
The
quadruplicate
replicate
runs
on
a
given
day
for
the
vehicle
control(
s)
and
androstenedione
(
highest
concentration
only)
will
be
used
for
quality
control
comparisons.
On
a
given
day,
the
batch
of
up
to
48
runs
will
include
half
of
the
quadruplicate
replicate
runs
at
the
beginning
and
half
at
the
end
for
the
vehicle
control
and
androstenedione.
A
two­
way
analysis
of
variance
will
be
carried
out
on
the
data.
This
same
comparison
will
be
made
for
the
androstenedione.
The
variation
between
the
two
replicate
runs
at
the
beginning
of
the
batch
and
the
two
replicate
runs
at
the
end
will
be
based
on
the
pooled
variation
across
batches.

By
definition
the
four
NSB
values
must
average
0
percent
aromatization
and
the
four
vehicle
values
must
average
100
percent
aromatization.
It
follows
that
for
these
endpoints,
a
comparison
can
be
made
for
the
average
of
the
first
two
values
to
0
percent
or
100
percent,
respectively.
If
the
test
conditions
remain
constant
across
the
duration
of
the
runs
within
a
batch,
then
the
NSB
average
within
each
batch
should
be
statistically
equivalent
to
0,
and
the
vehicle
average
within
each
batch
should
be
statistically
equivalent
to
100.
This
structure
will
be
examined
by
analysis
of
variance,
multiple
comparisons,
and
graphical
analysis.

For
each
of
the
responses,
the
standard
deviation
within
batches
will
be
compared
between
the
first
and
last
tests
and
across
batches
by
analysis
of
variance
and
graphical
techniques
in
a
manner
similar
to
the
average
values.

5.2.3
Concentration
Response
Curve
Fits
to
the
Standard
Tests
Competitive
concentration
response
curves
will
be
fitted
to
the
standard
substrate
test
results
within
each
batch.
The
NSB
runs
will
be
included
in
the
fits
but
the
vehicle
runs
will
not
be
included.
Responses
will
be
normalized
to
"
percent
aromatization",
based
on
the
NSB
values
and
the
"
corresponding
vehicle"
values.
Percent
aromatization
is
defined
as:

[
Observed
DPM
 
NSB
DPM]
/
[
Vehicle
DPM
 
NSB
DPM]
x
100
If
Y

percent
aromatization
and
x

log
10
(
concentration),
then
the
competitive
Battelle
Draft
13
June,
2002
concentration
response
relation
between
Y
and
x
can
be
described
by
the
model
Y
b
t
b
x
=
+
 
+
 
1
10
 
µ
(
)

where
t
and
b
are
the
"
top"
and
the
"
bottom"
of
the
curve
(
approximately
100%
and
0%
respectively),
 
is
the
slope,
and
µ
is
the
log
10
(
IC
50).
The
model
will
be
fitted
by
weighted
least
squares.
The
weights
are
inversely
proportional
to
the
predicted
amount
of
3H
2
O
as
measured
by
the
DPM
count.
Namely,

DPM
pred
NSB
DPM
(
)[
SPC
DPM]
Y
100
=
+

where
NSB
DPM
is
the
"
nonspecific
DPMs"
associated
with
the
NSB
concentration
of
standard
substrate
and
SPC
DPM
is
the
"
specific
DPMs",
i.
e.
vehicle
DPM
 
NSB
DPM:

Weight
K
DPM
pred
=

The
concentration
response
relation
will
be
fitted
within
each
test
batch.
Since
the
replicate
determinations
within
a
single
test
run
involve
only
replication
of
the
scintillation
counter
readings,
their
differences
do
not
reflect
all
of
the
variation.
Thus,
the
model
will
be
fitted
to
the
average
of
the
replicate
determinations
at
each
concentration.
This
process
will
be
repeated
in
each
test
batch.

Univariate
one
way
random
effects
analyses
of
variance
will
be
carried
out
on
the
results
of
the
fits.
The
responses
will
be
t,
b,
µ
,
 ,
std
err
µ
,
std
err
 .
The
standard
errors
are
the
within
batch
standard
errors.
Test
batch
will
be
treated
as
a
random
effect.
Batch­
to­
batch
variation
will
be
determined
for
each
of
the
parameters
and
will
be
tested
for
significance.
It
is
anticipated
that
the
batch­
to­
batch
variation
will
be
specific
to
the
assay.
If
the
batch­
to­
batch
variation
is
significant
it
will
be
incorporated
into
the
standard
error
of
the
corresponding
parameter
and
associated
confidence
intervals.

5.2.4
Concentration
Response
Curve
Fits
to
the
Test
Substances
Concentration
response
curves
will
be
fitted
to
the
results
from
each
test
substance
within
each
batch.
Plots
will
be
prepared
displaying
the
individual
percent
aromatization
determinations
and
the
concentration
response
curve
fits.
The
form
of
the
model
is
the
same
as
for
the
standard
tests,
namely
Y
b
t
b
x
=
+
 
+
 
1
10
 
µ
(
)

where
t
and
b
are
"
top"
and
the
"
bottom"
of
the
curve
(
approximately
100%
and
0%
respectively),
 
is
the
slope,
and
µ
is
the
log
10
(
IC
50).
The
"
top"
and
"
bottom"
parameters
t
and
b
Battelle
Draft
14
June,
2002
will
be
constrained
to
correspond
to
those
of
the
standard
curve
within
the
same
run,
resulting
in
a
two
parameter
model
fit.

The
model
will
be
fitted
by
weighted
least
squares.
The
weights
are
inversely
proportional
to
the
predicted
DPM
count.
Namely,

DPM
pred
NSB
DPM
(
)
[
SPC
DPM]
Y
100
=
+

where
NSB
DPM
is
the
"
nonspecific
DPM"
associated
with
the
NSB
concentration
of
standard
substance
and
SPC
DPM
is
the
"
specific
DPM",
i.
e.
vehicle
DPM
 
NSB
DPM.

Weight
K
DPM
pred
=

Based
on
the
results
of
the
fit
within
each
run
the
aromatase
inhibition
activity
will
be
expressed
as
the
IC
50
(
concentration
corresponding
to
50
percent
inhibition,
µ
)
.

The
concentration
response
relation
for
each
test
substance
will
be
fitted
within
four
independent
test
batch.
Since
the
replicate
determinations
within
a
test
run
involve
only
replication
of
scintillation
counter
readings
their
differences
do
not
reflect
all
of
the
within
run
variation.
Thus,
the
model
will
be
fitted
to
the
average
of
the
replicate
determinations
at
each
concentration.

Univariate
one
way
random
effects
analyses
of
variance
will
be
carried
out
on
log
10(
IC
50)
and
their
associated
within
batch
standard
errors.
Test
batches
will
be
treated
as
a
random
effect.
Batch­
to­
batch
variation
will
be
determined
for
each
of
the
parameters
and
will
be
tested
for
significance.
It
is
anticipated
that
the
batch­
to­
batch
variation
will
be
specific
to
the
test
substance
and
the
assay.
If
the
batch­
to­
batch
variation
is
significant
it
will
be
incorporated
into
the
standard
error
of
the
corresponding
parameter
and
associated
confidence
intervals.

5.2.5
Comparison
of
Optimized
Assays
For
each
test
compound
log
10
(
IC
50)
and
its
associated
standard
error,
degrees
of
freedom,
and
confidence
interval
will
be
determined
with
each
assay.
The
estimates
and
their
standard
errors
will
be
compared
among
assays
by
two­
sample
heterogeneous
variances
t­
tests,
adjusting
for
simultaneous
inferences.
Consistent
patterns
across
test
substances
of
inequalities
among
assays
will
be
examined.

5.2.6
Comparisons
Based
on
Reduced
Numbers
of
Test
Concentrations
The
comparisons
among
assays
discussed
above
were
based
on
IC
50
determinations
obtained
with
a
range
of
eight
concentrations
from
10­
9
M
to
10­
3
M.
These
comparisons
will
be
Battelle
Draft
15
June,
2002
repeated
with
a
subset
of
the
data,
with
a
range
of
three
concentrations
at
0.1,
1,
and
10

M
(
10­
7M
to
10­
5M).
Estimates
of
log
10
(
IC
50)
within
each
test
run
will
be
carried
out
as
if
the
available
data
were
limited
to:

°
test
concentrations
from
10­
7M
to
10­
5
M
for
each
test
substance
°
test
concentrations
from
10­
7M
to
10­
5
M
for
the
standard
substrate
°
NSB
concentration
of
10­
3
M
for
the
standard
substrate
°
the
vehicles.

The
determinations
of
the
IC
50
s
and
their
standard
errors
and
confidence
intervals
for
each
test
substance
will
be
repeated
in
the
manner
discussed
above,
but
based
only
on
the
available
data.
Comparisons
among
assays
will
be
carried
out
in
the
same
manner
as
discussed
above.

6.0
REFERENCES
Amarneh,
B.
and
Simpson,
E.
R.
Expression
of
a
recombinant
derivative
of
human
aromatase
P450
in
insect
cells
utilizing
the
baculovirus
vector
system.
Mol.
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