1. Sulindac Pharmacokinetics
The Role of Flavin-containing Monooxygenases
Brett Bemer
Dr. David Williams Laboratory
Dr. Sharon Krueger
Dr. Gayle Orner
HHMI Summer Research 2008

2. 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
Self-note: Sulindac is a not a salicylate, pyrazolone or propionic acid derivative
Sulindac
Aspirin

3. 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.
FAP: large number of colorectal adenomas at an early age – leads to colon cancer
Sulindac 200mg

4. Sulindac Activation/Inactivation
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)

5. Sulindac: Reduction (Activation)
Sulindac sulfoxide (prodrug) is reduced to sulindac sulfide (active) in the gut
Sulindac sulfoxide
Sulindac sulfide

6. Sulindac: Oxidation (Inactivation)
Sulindac sulfide (active) is reversibly reoxidized back to the sulfoxide (prodrug) in the liver
Sulindac sulfide
Sulindac sulfoxide

7. Sulindac: Oxidation (Inactivation)
Sulindac sulfoxide (prodrug) is then irreversibly oxidized a second time to sulindac sulfone (inactive)
Sulindac sulfoxide
Sulindac sulfone

8. 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.

9. 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
I could add that if FMO3 activity is completely/mostly lost (as it is with some mutations), fish odor syndrome results.

10. FMO3: Polymorphism Frequency
FMO3 mutation frequency (in white populations):
E158K: 0.426
E308G: 0.225
V257M: 0.069
Self-note: As Kevin pointed out, I need to remember to explain allelic frequency and it’s significance. Also note that many other polymorphisms exist as well.
Sachse et. al. Pharmacogenetics and Genomics,1999

11. Indole-3-carbinol
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

12. 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

13. 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.

14. The Diet Study
Human subjects ingest sulindac following dietary intervention
The 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 mg 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

15. 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 Waters HPLC
Typical chromatogram of FMO3 incubation with SS

16. Experiment: Kinetic Assays
FMO3 proteins incubated with sulindac sulfide in the presence of NADPH
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
Clarify at some point that our laboratory had generated over-expressed FMO3 and variants from cloned cDNA

17. Experiment: Kinetic Assays
Determination of Km, Vmax, and kcat values
Characterizes protein’s affinity for Sulindac as a substrate
Vmax – Maximum velocity
Kcat – describes catalytic rate, enzymatic reactions per second
Km—substrate concentration at half maximal velocity
A typical Lineweaver-Burk plot

18. 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
Not that FMO2 polymorphisms are also being tested for.
Presence or absence of a polymorphism results in variable digestion products

19. Wildtype Allele Band Sizes Mutant Allele Band Sizes
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 7
E305X
E308G
EcoRI
ApaI
165/33
198
174/24 6
V257M
BsaAI
197/132
329
aPrimer pairs from Dolphin et al., 1997 Nat Genet 17:491-4.
bPrimer pairs from Sachse et al., 1999 Clin Pharmacol Therap 66:431-8.
cPrimer pairs from Dolphin et al., 2000 Pharmacogenetics 10:

20. Genotyping: E158K Example
Wildtype-230bp E158K-284bp

21. 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

22. Where We Are Going
Human samples must be collected, extracted, and analyzed
First individual completed both diets and samples are in storage
9-14 additional individuals will proceed through study over the next several months
Following data collection…
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

23. Acknowledgements Dr. Sharon Krueger & Dr. Gayle Orner
Dr. Williams Laboratory
HHMI
USANA, NIH, URISC
LPI
Dr. Kevin Ahern