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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. Nonsteroidal anti-inflammatory drug (NSAID) available as Clinoril

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Sulindac Pharmacokinetics

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  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 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. Sulindac 200mg

  4. 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)

  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

  10. 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

  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 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

  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

  17. Experiment: Kinetic Assays • Determination of Km, Vmax, and kcat values • Characterizes protein’s affinity for Sulindac as a substrate 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

  19. 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.

  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

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