Transcript Warfarin - Tryptophan.net

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 γcarboxyGlu
 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 Xtargeting 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
Severe
Reached
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
Amino acid
area
substitutions
No. of
samples
Berlin
E37G
12
Lower Saxony
E37G
1
R58G
13
Westphalia
R12W, A26S, A48T,
R58G, R61L
R12W, A26S, A48T, R61L
Rhineland
Azores
L128S
7
2
17
Y139C
1
Y139C
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?