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Transcript of Sulindac Pharmacokinetics The Role of Flavin-containing Monooxygenases Brett Bemer Dr. David...
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
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: 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
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 sulfoxide (prodrug) is reduced to sulindac sulfide (active) in the gut
Sulindac sulfoxide Sulindac sulfide
Sulindac: Oxidation (Inactivation)
Sulindac sulfide (active) is reversibly reoxidized back to the sulfoxide (prodrug) in the liver
Sulindac sulfoxideSulindac sulfide
Sulindac: Oxidation (Inactivation)
Sulindac sulfoxide (prodrug) is then irreversibly oxidized a second time to sulindac sulfone (inactive)
Sulindac sulfoxide Sulindac sulfone
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 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
Sachse et. al. Pharmacogenetics and Genomics,1999
FMO3 mutation frequency (in white populations):
E158K: 0.426
E308G: 0.225
V257M: 0.069
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.
Indole-3-carbinol
Broccoli
Brussels sprouts
Cauliflower
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
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 StudyHuman 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
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
AU
0.00
0.20
0.40
0.60
0.80
1.00
Minutes5.00 10.00 15.00
SOX
SS
Typical chromatogram of FMO3 incubation with SS
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 Assays
Determination of Km, Vmax, and kcat values
Characterizes protein’s affinity for Sulindac as a substrate
Lineweaver-Burk
y = 3.306x + 0.0343R2 = 0.9892
0.000
0.050
0.100
0.150
0.200
0.250
-0.020 0.000 0.020 0.040
1/[S]
1/V
A typical Lineweaver-Burk plot
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 StrategyExpected band sizes for polymorphism detection
Exon Mutants detected
Restriction Enzyme
Wildtype Allele Band
Sizes
Mutant Allele Band Sizes
4 P153LE158K
BamHIHinfI
248/36230/54
284284
7 E305XE308G
EcoRIApaI
165/33198
198174/24
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 10:799-807.
Genotyping: E158K Example
Wildtype-230bp E158K-284bp
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 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
Dr. Sharon Krueger & Dr. Gayle Orner Dr. Williams Laboratory HHMI USANA, NIH, URISC LPI Dr. Kevin Ahern