The development and application of a surface plasmon ... - DCU

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B. Fitzpatrick, R. O’Kennedy / Journal of Immunological Methods 291 (2004) 11–25
and his team synthesised over 100 4-hydroxycoumarin-type
compounds and found that the minimal
structural requirements for anticoagulant activity
were an intact 4-hydroxycoumarin residue with the
3-position substituted by a carbon residue. Warfarin
was subsequently synthesised (Ikawa et al., 1944)
and is currently the most widely prescribed oral
anticoagulant for the management of a variety of
thromboembolic disorders including atrial fibrillation,
deep vein thrombosis and threatened stroke
(Hirsh et al., 1995).
Warfarin exerts its anticoagulant effect by inhibiting
the posttranslational carboxylation of the vitamin
K-dependent precursor proteins factors II, VII, IX
and X, resulting in the production of partially carboxylated
forms of these vitamin K-dependent proteins.
The presence of these partially carboxylated
proteins, named PIVKAs (proteins induced by vitamin
K antagonism), results in a slower activation of
the coagulation cascade, as the g-carboxyglutamyl
residues confer unique metal-binding properties to
the proteins. Upon occupancy of these metal-binding
sites, these proteins undergo a conformational
change, which permit interactions with the phospholipid
bilayers and cell membrane providing a template
for the clotting mechanism (Cooke et al.,
1997).
Warfarin is administered orally as a racemic mixture
of (R-) and (S-) warfarin and is rapidly absorbed from
the gastrointestinal tract reaching peak plasma concentrations
within 60–90 min following administration
(King et al., 1995). Following absorption, the drug is
highly bound to plasma proteins (>99%), leaving only
a small fraction of the free drug available for metabolism.
It is the ‘nonprotein’-bound or ‘nonprotein’-free
fraction of the administered drug that dictates its
pharmacokinetic profile. The primary warfarin-binding
site on human serum albumin (HSA) has been identified
on domain II with a secondary binding site on
domain I (Dockal et al., 1999). The (S-) enantiomer has
been shown to have a higher affinity for HSA than the
corresponding (R-) enantiomer (Bertucci et al., 1999).
This high degree of protein binding predisposes warfarin
to a range of possible drug interactions, which in
turn can greatly affect the degree of anticoagulation
achieved (Wells et al., 1994). Moreover, the (S-)
enantiomer has also approximately five times the
anticoagulant potency of the (R-) enantiomer.
Warfarin has also shown promising results as a
potent HIV-1 inhibitor in vitro, and this has led to the
development of a potent orally bioavailable class of
5,6-dihydro-4-hydroxy-2-pyrones compounds, which
are undergoing clinical trials (Thaisrivongs and Strohbach,
1999). Warfarin has also demonstrated potential
as an antimetastatic agent and has shown particularly
promising results with small cell carcinoma of the
lung, a tumour cell type that is characterised by a
coagulation-associated pathway (Zacharski et al.,
1984; Amirkhosravi and Francis, 1995).
There is currently a wide range of analytical
techniques available for the determination of warfarin
in biological fluids ranging from chromatography to
phosphorescence-based measurements (Capitán-Vallvey
et al., 1999; Ring and Bostick, 2000). The
majority of these analytical techniques involve
lengthy sample pretreatment, derivatisation schemes
and postcolumn reactions for the determination of the
free ‘unbound’ fraction of warfarin in plasma samples
and, consequently, are not routinely applicable for the
detection of warfarin in clinical samples. The use of
chromatographic techniques relies on the inherent
physicochemical properties of the analyte in question,
and the measured response is usually directly proportional
to the concentration of the analyte and is best
represented by a linear calibration model. The physicochemical
properties of a given analyte and the lack
of suitable chromophoric or electrochemically active
groups can therefore be limiting factors for the detailed
analysis of the pharmacokinetic profile of
particular drug molecules. Immunoassays rely on the
specific interaction between antibody and antigen for
analyte determination, and the measured response
usually gives rise to a calibration plot that is inherently
sigmoidal in nature and best represented by a
four-parameter logistic fit (Findlay et al., 2000).
Recent advances in antibody technology have permitted
the standardisation of antibody preparations and
revolutionised their use as clinical and diagnostic
tools. The capability of producing antibodies and
fragments thereof with enhanced affinities (i.e.,
K D =10
15 M; Boder et al., 2000) allied with developments
in transduction technologies have greatly
advanced the potential of antibody-based techniques
for the detection of low molecular weight analytes in
solution. Similarly, given the unique specificity of
antibodies and the use of antibody libraries, common