Fluorescence enhancement of warfarin induced by interaction with β‐cyclodextrin

Warfarin is the most common agent used for control and prevention of venous as well as arterial thromboembolism (blood clots). In aqueous media, warfarin forms inclusion complexes with a family of cyclic oligosaccharides, α, β, γ‐cyclodextrins (CD). The formation of these complexes results in enhancement of the fluorescence of warfarin. Such spectroscopic changes offer a venue for the development of bioanalytical methodologies for warfarin quantification in biological liquids. We characterized the photophysical properties of warfarin in solvents with varying polarity and viscosity. The fluorescence quantum yield of warfarin correlated: (1) strongly with the solvent viscosity (R = 0.979) and (2) weakly with the solvent polarity (R = 0.118). These findings indicate that it is the change of the viscosity, rather than polarity, of the microenvironment that causes the fluorescence enhancement of warfarin upon binding to β‐CD. Utilizing the observed fluorescence enhancement in fluorescence titration measurements, the binding constants of warfarin to β‐CD were obtained (2.6 × 10² M^?1–3.7 × 10² M^?1). Using multivariable linear analysis, we extracted the stoichiometry of warfarin‐β‐CD interaction (1:1). © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Catheter Ablation in Combination With Left Atrial Appendage Closure for Atrial Fibrillation

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, affecting millions of individuals worldwide ^1-3. The rapid, irregular, and disordered electrical activity in the atria gives rise to palpitations, fatigue, dyspnea, chest pain and dizziness with or without syncope ^{4, 5}. Patients with AF have a five-fold higher risk of stroke ⁶.Oral anticoagulation (OAC) with warfarin is commonly used for stroke prevention in patients with AF and has been shown to reduce the risk of stroke by 64% ⁷. Warfarin therapy has several major disadvantages, however, including bleeding, non-tolerance, interactions with other medications and foods, non-compliance and a narrow therapeutic range ^8-11. These issues, together with poor appreciation of the risk-benefit ratio, unawareness of guidelines, or absence of an OAC monitoring outpatient clinic may explain why only 30-60% of patients with AF are prescribed this drug ⁸.The problems associated with warfarin, combined with the limited efficacy and/or serious side effects associated with other medications used for AF ^12,13, highlight the need for effective non-pharmacological approaches to treatment. One such approach is catheter ablation (CA), a procedure in which a radiofrequency electrical current is applied to regions of the heart to create small ablation lesions that electrically isolate potential AF triggers ⁴. CA is a well-established treatment for AF symptoms ^{14, 15}, that may also decrease the risk of stroke. Recent data showed a significant decrease in the relative risk of stroke and transient ischemic attack events among patients who underwent ablation compared with those undergoing antiarrhythmic drug therapy ¹⁶.Since the left atrial appendage (LAA) is the source of thrombi in more than 90% of patients with non-valvular atrial fibrillation ¹⁷, another approach to stroke prevention is to physically block clots from exiting the LAA. One method for occluding the LAA is via percutaneous placement of the WATCHMAN LAA closure device. The WATCHMAN device resembles a small parachute. It consists of a nitinol frame covered by fabric polyethyl terephthalate that prevents emboli, but not blood, from exiting during the healing process. Fixation anchors around the perimeter secure the device in the LAA (Figure 1). To date, the WATCHMAN is the only implanted percutaneous device for which a randomized clinical trial has been reported. In this study, implantation of the WATCHMAN was found to be at least as effective as warfarin in preventing stroke (all-causes) and death (all-causes) ¹⁸. This device received the Conformité Européenne (CE) mark for use in the European Union for warfarin eligible patients and in those who have a contraindication to anticoagulation therapy ¹⁹.Given the proven effectiveness of CA to alleviate AF symptoms and the promising data with regard to reduction of thromboembolic events with both CA and WATCHMAN implantation, combining the two procedures is hoped to further reduce the incidence of stroke in high-risk patients while simultaneously relieving symptoms. The combined procedure may eventually enable patients to undergo implantation of the WATCHMAN device without subsequent warfarin treatment, since the CA procedure itself reduces thromboembolic events. This would present an avenue of treatment previously unavailable to patients ineligible for warfarin treatment because of recurrent bleeding ²⁰ or other warfarin-associated problems.The combined procedure is performed under general anesthesia with biplane fluoroscopy and TEE guidance. Catheter ablation is followed by implantation of the WATCHMAN LAA closure device. Data from a non-randomized trial with 10 patients demonstrates that this procedure can be safely performed in patients with a CHADS₂ score of greater than 1 ²¹. Further studies to examine the effectiveness of the combined procedure in reducing symptoms from AF and associated stroke are therefore warranted.     

Probing the structure of the warfarin-binding site on human serum albumin using site-directed mutagenesis

The binding of warfarin to the following human serum albumin (HSA) mutants was examined: K195M, K199M, F211V, W214L, R218M, R222M, H242V, and R257M. Warfarin bound to human serum albumin (HSA) exhibits an intrinsic fluorescence that is approximately 10-fold greater than the corresponding signal for warfarin in aqueous solution. This property of the warfarin/HSA complex has been widely used to determine the dissociation constant for the interaction. In the present study, such a technique was used to show that specific substitutions in subdomain 2A altered the affinity of HSA for warfarin. The fluorescence of warfarin/mutant HSA complexes varied widely from the fluorescence of the warfarin/wild-type HSA complex at pH = 7.4, suggesting changes in the structure of the complex resulting from specific substitutions. The fluorescence of the warfarin/wild-type HSA complex increases about twofold as the pH is increased from 6.0 to 9.0 due to the neutral-to-base (N-B) transition, a conformational change that occurs in HSA as a function of pH. Changes in the fluorescence of warfarin/mutant HSA complexes as a function of pH suggests novel behavior for most HSA species examined. For the HSA mutants F211V and H242V, the midpoint of the N-B transition shifts from a wild-type pH of 7.8 to a pH value of 7.1-7.2.     

Benzoates of cellulose bonded on silica gel: Chiral discrimination ability as high-performance liquid chromatographic chiral stationary phases

In order to generalize the recently described method for the insolubilization of polysaccharide derivatives to benzoates of cellulose, five mixed 10-undecenoate/benzoates of this polysaccharide have been prepared and linked to allyl silica gel by means of a radical reaction. The chiral recognition ability of the resulting materials when used as high-performance liquid chromatographic chiral stationary phases was evaluated using heptane/2-propanol and heptane/chloroform mixtures as mobile phases. Chirality 9:145–149, 1997. © 1997 Wiley-Liss, Inc.     

Carbamates of cellulose bonded on silica gel: Chiral discrimination ability as HPLC chiral stationary phases

Four cellulose mixed 10-undecenoate/carbamate derivatives, simultaneously bearing 10-undecenoyl and variously substituted phenylaminocarbonyl groups, were chemically bonded on allylsilica gel. The study of the effect of these substitutions on the performance of the resulting chiral supports, and a comparison with the recently described 10-undecenoate/3,5-dimethylphenylcarbamate derivative, are presented. In this study heptane/2-propanol or heptane/chloroform mixtures were used as mobile phases. Chirality 10:283–288, 1998. © 1998 Wiley-Liss, Inc.     

The separation of bovine prothrombin and descarboxyprothrombin by high-performance liquid chromatography

Prothrombin contains 10 γ-carboxyglutamic acid (Gla) residues which are absent in the warfarin-induced descarboxyprothrombin; hence prothrombin has 10 more negative groups than has descarboxyprothrombin. The two proteins can be separated by HPLC with the aid of an anion-exchange column. Plasma from warfarin-treated animals could be analyzed without pretreatment of the samples and a full analysis was obtained in 30 min.     

Warfarin metabolites: Stereochemical aspects of protein binding and displacement by phenylbutazone

The in vitro human serum albumin binding characteristics of the enantiomers of the major metabolites of warfarin [6-hydroxywarfarin (6-HW), 7-hydroxywarfarin (7-HW), (S)-warfarin alcohols [(S,S)- and (S,R)-WA], and (R,S)-warfarin alcohol [(R,S)-WA]] have been studied, using a stereospecific HPLC assay. Warfarin metabolites are less bound both within plasma and a 40 g/liter solution of human serum albumin than the enantiomers of warfarin. The reduced warfarin metabolites have a lower fraction unbound [1.33% for (S,R)-WA, 2.09% for (S,S)-WA, and 1.04% for (R,S)-WA] than hydroxylated metabolites [3.24% for (R)-6-HW, 4.26% (S)-6-HW, 4.49% for (R)-7-HW and 4.27% for (S)-7-HW] to HSA. Phenylbutazone produced a concentration-dependent increase in the unbound fraction of all metabolites. It was possible to predict the unbound fraction of warfarin metabolites based on the unbound fraction of warfarin enantiomers. © 1993 Wiley-Liss, Inc.     

Home range and movement in populations of Rattus norvegicus polymorphic ior warfarin resistance

Movements of brown rats were deduced from records made during a capture-recapture study of rural populations on two farms in Mid-Wales. Populations in the hedges were sampled at monthly intervals for two years, but samples from the farm buildings were obtained only irregularly and by a variety of methods.
On the main trap line, of the rats captured during at least two sampling periods 35 (78%) females and 57 (74%) males appeared to have established home ranges, the best estimates of mean home range length for each sex being 54.8 m and 66.1 m respectively. The longest recorded distances travelled during known life were 850 m for a female and 954 m for a male, although the median distances travelled were only 43 m and 52 m respectively. The median distance travelled during a sampling period (seven nights) was about 24 m for both sexes. There were no significant differences between the distances travelled by the sexes or by different age groups, and there was little seasonal variation.
Infestations in the hedges were always associated with streams, and those more than about 100 m from the buildings seldom persisted through winter. During autumn and winter the rats in the more distant localities tended to move towards the buildings, but there was no suggestion of an orderly migration. Twenty-seven out of 386 rats marked in the hedges were recovered amongst 576 taken from the buildings, but only nine of diem had travelled more than 100 m. No rats released in the buildings were captured in the hedges. Rats established in the buildings seemed to exclude rats immigrating from the hedges.