Sulindac Pharmacokinetics

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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
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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
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Sulindac Background

• Shown to exhibit chemopreventative properties
• Effective in reducing adenomas in familial
  adenomatous polyposis (FAP) patients
• However, sulindacs effectiveness is
  substantially inhibited over time due to drug
  resistance and metabolic inactivation.

Sulindac 200mg
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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)

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Sulindac Reduction (Activation)

• Sulindac sulfoxide (prodrug) is reduced to
  sulindac sulfide (active) in the gut

Sulindac sulfoxide
Sulindac sulfide
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Sulindac Oxidation (Inactivation)

• Sulindac sulfide (active) is reversibly
  reoxidized back to the sulfoxide (prodrug) in the
  liver

Sulindac sulfoxide
Sulindac sulfide
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Sulindac Oxidation (Inactivation)

• Sulindac sulfoxide (prodrug) is then irreversibly
  oxidized a second time to sulindac sulfone
  (inactive)

Sulindac sulfoxide
Sulindac sulfone
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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.

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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

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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
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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
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Summary of Observations

• Sulindac is a potentially effective anti-cancer
  agent
• Sulindacs 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

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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.

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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

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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
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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

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Experiment Kinetic Assays

• Determination of Km, Vmax, and kcat values
• Characterizes proteins affinity for Sulindac as
  a substrate

A typical Lineweaver-Burk plot
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Genotyping Strategy

• Employment of polymerase chain reaction-restrictio
  n 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

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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
17491-4. bPrimer pairs from Sachse et al., 1999
Clin Pharmacol Therap 66431-8. cPrimer pairs
from Dolphin et al., 2000 Pharmacogenetics
10799-807.
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Genotyping E158K Example

• Wildtype-230bp E158K-284bp

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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

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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

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Acknowledgements

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