Transcript Sulindac Pharmacokinetics The Role of Flavin-containing Monooxygenases Brett Bemer Dr. David Williams Laboratory Dr.

Sulindac Pharmacokinetics
The Role of Flavin-containing Monooxygenases
Brett Bemer
Dr. David Williams Laboratory
Dr. Sharon Krueger
Dr. Gayle Orner
HHMI Summer Research 2008
Sulindac: Background

Nonsteroidal anti-inflammatory drug (NSAID) available as Clinoril

NSAIDs are effective in treating pain, fever, and inflammation


Clinoril itself is normally prescribed for relieving pain associated with
rheumatoid arthritis
Other NSAIDs include aspirin and ibuprofen
Sulindac
Aspirin
Sulindac: Background

Shown to exhibit chemopreventative properties


Effective in reducing adenomas in familial adenomatous
polyposis (FAP) patients
However, sulindac’s effectiveness is substantially inhibited
over time due to drug resistance and metabolic inactivation.
Sulindac 200mg
Sulindac Activation/Inactivation

Activation:


Sulindac sulfoxide (prodrug) is reduced to sulindac sulfide (active) in the
gut
Inactivation:

Sulindac sulfide (active) is reversibly reoxidized back to the sulfoxide
(prodrug) in the liver

Sulindac sulfoxide (prodrug) is then irreversibly oxidized a second time
to sulindac sulfone (inactive)
Sulindac: Reduction (Activation)

Sulindac sulfoxide (prodrug) is reduced to sulindac sulfide (active) in
the gut
Sulindac sulfoxide
Sulindac sulfide
Sulindac: Oxidation (Inactivation)

Sulindac sulfide (active) is reversibly reoxidized back to the sulfoxide
(prodrug) in the liver
Sulindac sulfide
Sulindac sulfoxide
Sulindac: Oxidation (Inactivation)

Sulindac sulfoxide (prodrug) is then irreversibly oxidized a second
time to sulindac sulfone (inactive)
Sulindac sulfoxide
Sulindac sulfone
FMO: Background

Flavin-containing monooxygenase (FMO) protein family

Family of proteins that catalyze oxidation reactions with the
cofactor flavin adenine dinucleotide

Known for catalyzing oxidations of a wide variety of xenobiotics,
and endogenous substrates.

Known particularly for catalyzing oxidation of compounds
containing sulfur and nitrogen groups that are susceptible to
oxidation.
FMO3: Background

The enzyme primarily responsible for Sulindac inactivation is
FMO3 (FMO isoform 3)

Many known FMO3 polymorphisms exist


Polymorphic FMO3 proteins can exhibit reduced enzymatic activity for
a wide range of substrates
Two common polymorphisms, E158K and E308G (SNPs), have
been shown to occur more frequently in FAP patients that
respond well to Sulindac
FMO3: Polymorphism Frequency

FMO3 mutation frequency (in white populations):
E158K:
0.426
E308G:
0.225
V257M:
0.069
Sachse et. al. Pharmacogenetics and Genomics,1999
Indole-3-carbinol
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In addition, FMO activity has been shown to be strongly inhibited by
indole-3-carbinol.

Indole-3-carbinol: An indole derivative that is found at high levels in
cruciferous vegetables.
Cauliflower
Broccoli
Indole-3-carbinol
Brussels sprouts
Summary of Observations

Sulindac is a potentially effective anti-cancer agent

Sulindac’s effectiveness is reduced when it is oxidized and
inactivated by FMO3

FMO3 polymorphisms E158K and E308G have been shown to occur
more frequently in FAP patients that respond well to Sulindac.

In addition, dietary indoles, particularly indole-3-carbinol, have been
shown to inhibit FMO3 activity
Predictions

FMO3 polymorphisms E158K and E308G will produce
proteins that exhibit lower affinity for sulindac sulfide than the
wildtype FMO3 protein


Analysis performed by obtaining in vitro kinetics via HPLC
Human subjects following an indole-3-carbinol rich diet will
inactivate less sulindac than the same subjects on a low/no
indole diet.

Blood draws taken during a time course will be analyzed for
Sulindac levels.
The Diet Study
Human
The
subjects ingest sulindac following dietary intervention
diet:
Participants
take part in a two week washout period (no cruciferous vegetables)
Participants
take part in two week diet; half ingesting 300 grams of Brussels
sprouts/day, half ingesting 0 grams
On
day 28 200mg of Sulindac is administered and blood draws taken at 0, 1, 2, 3, 4, 5,
6, 7, 8, 24, and 48 hours
Procedure
repeats, but the participants who ingested 300 grams Brussels sprouts will
ingest 0 grams, and vice versa
Quantification of Sulindac Levels

In vivo metabolism of Sulindac is analyzed by extraction of
Sulindac (parent and products) from collected blood and
detection on a Waters HPLC.

Sulindac products extracted into 1-chlorobutane fractions,
dried, and redissolved in 100µl mobile phase

Sulindac products quantified by detection at 330nm on a
1.00
Waters HPLC
0.80
0.60
U
A
X
O
S
S
S
0.40
0.20
0.00
5.00
10.00
15.00
Minutes
Typical chromatogram of FMO3 incubation with SS
Experiment: Kinetic Assays

FMO3 proteins incubated with sulindac sulfide in the presence of
NADPH

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Substrate concentrations range from 5µM to 200µM
Sulindac products extracted into ethyl acetate fractions, dried,
redissolved in 100µl mobile phase, and detected at 330nm on a
Waters HPLC
Experiment: Kinetic Assays
Determination of Km, Vmax, and kcat values

Characterizes protein’s affinity for Sulindac as a substrate
Linew eaver-Burk
0.250
0.200
0.150
1/V

y = 3.306x + 0.0343
R2 = 0.9892
0.100
0.050
-0.020
0.000
0.000
0.020
1/[S]
A typical Lineweaver-Burk plot
0.040
Genotyping Strategy

Employment of polymerase chain reaction-restriction fragment length
polymorphism (PCR-RFLP)




1) DNA extracted from anti-coagulated blood samples
2) DNA from exons 4 and 7 amplified by PCR
3) Assay for SNPs via restriction enzyme digest of products
4) Bands separated and via gel electrophoresis
Genotyping Strategy
Expected band sizes for polymorphism detection
Exon
Mutants
detected
Restriction
Enzyme
Wildtype
Allele Band
Sizes
Mutant
Allele
Band
Sizes
4
P153L
E158K
BamHI
HinfI
248/36
230/54
284
284
7
E305X
E308G
EcoRI
ApaI
165/33
198
198
174/24
6
V257M
BsaAI
197/132
329
aPrimer
pairs from Dolphin et al., 1997 Nat Genet 17:491-4.
pairs from Sachse et al., 1999 Clin Pharmacol Therap 66:431-8.
cPrimer pairs from Dolphin et al., 2000 Pharmacogenetics 10:799-807.
bPrimer
Genotyping: E158K Example

Wildtype-230bp
E158K-284bp
Where We Stand Now







Verify extraction methods from blood
Determine PCR methods that gave clean products for FMO3
Verify published PCR methods for FMO2 polymorphism detection
Verify that published methods (primers and digests) are working
Completed HPLC workup (extraction methods, solvent selection, etc.)
Determined conditions for over-expressed variant protein incubations
Determine kinetics for over-expressed variant proteins

Currently repeating reference protein and have yet to do two more
variants
Where We Are Going

Human samples must be collected, extracted, and analyzed



Following data collection…




First individual completed both diets and samples are in storage
9-14 additional individuals will proceed through study over the
next several months
Correlate sulindac parent/metabolite levels in blood with diet
Correlate sulindac parent/metabolite levels with genotype
Verify kinetics information
If results match predictions, apply dietary intervention with
sulindac in FAP patients to enhance outcome of sulindac
treatment
Acknowledgements




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
Dr. Sharon Krueger & Dr. Gayle Orner
Dr. Williams Laboratory
HHMI
USANA, NIH, URISC
LPI
Dr. Kevin Ahern