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