Pharmacogenomics

Case study 1: Warfarin

Warfarin overview

Warfarin is an anticoagulant drug which inhibits vitamin K 2,3-epoxide reductase.

Warfarin is used to reduce blood clots and after stroke. The therapeutic index (=LD50/ED50) for warfarin is small.

Warfarin is a coumarin, a derviative of dicoumarol, which was discovered in spoiled sweet clover.

It was developed by the University of Wisconsin. The name is derived from Wisconsin Alumni Research

Foundation coumarin. Similar drugs are acenocoumarol and phenprocoumon.

Warfarin action is affected by both pharmacodynamics and pharmacokinetics

Variations in Vitamin K epoxide reductase could result in more or less inhibition by warfarin.

Variations in vitamin K dependent carboxylase could also affect warfarin activity.

Variability in CYP450 2C9 and 3A4 will affect metabolism.

The Vitamin K cycle

Vitamin K is required for formation of γ-carboxyGlu

A number of proteins are known to contain γ-carboxyGlu residues.

These include blood coagulation factors II (prothrombin), VII, IX, and X, the anticoagulant proteins C and S, and the Factor X-targeting protein Z.

This modification is necessary for blood clotting. γ-CarboxyGlu is made in a post-translational modification

requiring vitamin K.

Vitamin Ks are naphthoquinones

The proposed mechanism of vitamin K dependent carboxylase

Does Vitamin K dependent Glu carboxylase affect warfarin response ?

A number of polymorphisms of GGCX have been identified. Most of these are in the non-coding regions. One mutation, R325Q, is found in the coding region. R325Q has about 30% higher activity than “wild-type”. These mutations were found to have only a modest effect (6-

10%) on the warfarin dose. No crystal structure of this endoplasmic reticulum associated

protein has been obtained.

Question?

Vitamin K dependent Glu carboxylase is not the target of warfarin. Why could it affect warfarin activity?

Recycling of vitamin K

After carboxylation, vitamin K is in the oxidized epoxide form.

It needs to be reduced back to the hydroquinone for the next cycle of carboxylation.

This is done by vitamin K 2,3-epoxide reductase. This enzyme is the target of warfarin inhibition.

A bacterial analogue structure of vitamin K reductase

The quinone binding site

The proposed reduction sequence

A possible mechanism for vitamin K epoxide reduction

Polymorphisms in VKOR

Polymorphism Daily Warfarin Dose mg

Resistance Phenotype

Wild type 4–6

A41S 16 Moderate

R58G 34 Major

V66M 31 Major

L28R >45 Severe

V45A Target INR never

Reached

Severe

Common SNPs in

noncoding regions

1–15 Variations across the

“normal” dosing range

Many of these mutations are at or near the quinone binding site

Location of mutations known to affect warfarin binding

Stereochemistry of warfarin inhibition of VKOR

The activity of the two enantiomers towards VKOR differs, with S-warfarin being 3–5 times more potent than R-warfarin.

Warfarin is a racemic mixture. Thus, S-Warfarin accounts for approximately 70% of

the overall anticoagulant activity.

Warfarin resistance has also appeared in animals

Warfarin has been used as rodent poison for about 50 years.

However, resistance has developed in rats and mice. These rodents show polymorphism in VKOR. Resistance may be due at least in part to VKOR

polymorphisms.

Mouse polymorphisms in VKOR

Geographic

area

Amino acid

substitutionsNo. of samples

Berlin E37G 12

Lower Saxony E37G 1

Westphalia R58G 13

R12W, A26S, A48T, R58G, R61L 7

R12W, A26S, A48T, R61L 2

Rhineland L128S 17

Y139C 1

AzoresY139C 1

How do the mutations in rats and mice compare with humans?

Most of the the mutations in rats and mice are different than those is humans.

However, the mutations occur in the same regions of the protein, positions 29 to 48 in exon 1, positions 58 to 67 in exon 2, and 120 to 143 in exon 3.

It is unclear if the mutations in VKOR are solely responsible for resistance in rodents.

Most of the mutations result in reduced enzyme activity.

Question?

Why do all of the mutations found in VKOR result in weaker rather than stronger warfarin binding?

What other enzyme(s) could contribute to warfarin resistance?

Question?

What are some other drugs that should not be used with warfarin?