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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

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sulindac pharmacokinetics

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
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 background3
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
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: Reduction (Activation)
  • Sulindac sulfoxide (prodrug) is reduced to sulindac sulfide (active) in the gut

Sulindac sulfoxide

Sulindac sulfide

sulindac oxidation inactivation
Sulindac: Oxidation (Inactivation)
  • Sulindac sulfide (active) is reversibly reoxidized back to the sulfoxide (prodrug) in the liver

Sulindac sulfide

Sulindac sulfoxide

sulindac oxidation inactivation7
Sulindac: Oxidation (Inactivation)
  • Sulindac sulfoxide (prodrug) is then irreversibly oxidized a second time to sulindac sulfone (inactive)

Sulindac sulfoxide

Sulindac sulfone

fmo background
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
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: 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
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

summary of observations
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
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
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 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
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

experiment kinetic assays
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
experiment kinetic assays17
Experiment: Kinetic Assays
  • Determination of Km, Vmax, and kcat values
    • Characterizes protein’s affinity for Sulindac as a substrate

A typical Lineweaver-Burk plot

genotyping strategy
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 strategy19
Genotyping Strategy

Expected band sizes for polymorphism detection

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 10:799-807.

genotyping e158k example
Genotyping: E158K Example
  • Wildtype-230bp E158K-284bp
where we stand now
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
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
acknowledgements
Acknowledgements
  • Dr. Sharon Krueger & Dr. Gayle Orner
  • Dr. Williams Laboratory
  • HHMI
  • USANA, NIH, URISC
  • LPI
  • Dr. Kevin Ahern
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