1 / 23

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

art
Télécharger la présentation

Sulindac Pharmacokinetics

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related