Interaction study between antiplatelet agents, anticoagulants, thyroid replacement therapy and a bioavailable formulation of curcumin (Meriva®).

OBJECTIVE: The objective of this clinical study is to evaluate possible interactions between antiplatelet agents, anticoagulants, thyroid hormone replacement therapy and a formulation of curcumin (Meriva®) that resulted effective for the complementary treatment of osteoarthritis. PATIENTS AND METHODS: Interaction between antiplatelet agents and Meriva® was evaluated by measuring anti-platelet activity with the in-vivo bleeding-time (BT) in patients assuming acetylsalicylic acid or ticlopidine or clopidogrel from at least 2 years. The BT was evaluated before and after 10 days of supplementation with Meriva®. The interaction between anticoagulants and Meriva® was evaluated in patients using warfarin or dabigatran for previous venous thrombosis. The INR level was evaluated before and after 10 days of supplementation with the curcumin formulation. Thyroid function tests in hypothyroid patients using LT4 replacement therapy (Eutirox®) were evaluated before and after 15 days of supplementation with Meriva®. Similarly, levels of glycemia and glycated hemoglobin were evaluated in diabetic patients in treatment with metformin, before and after 10 days of supplementation with the studied product. RESULTS: After 10 days of supplementation with Meriva® the average BT value was not significantly different for patients assuming acetylsalicylic acid, ticlopidine or clopidogrel at standard dosages. Similarly, after 10 days of Meriva® treatment, the INR level in the two groups of patients assuming warfarin or dabigatran was not statistically different from that observed at baseline. In the analyzed patients assuming LT4 or metformin, no interactions between the therapy and Meriva® were observed. CONCLUSIONS: Results from this non-interaction clinical study suggest that Meriva® does not interfere with the antiplatelet activity of the most common antiplatelet agents nor alters the INR values in stable patients assuming warfarin or dabigatran. Similarly, dosages of LT4 or metformin do not need to be adjusted in case of complementary treatment with Meriva®.

Discrete SeNPs-Macromolecule Binding Manipulated by Hydrophilic Interaction.

Nanoparticle-protein conjugates are promising probes for biological diagnostics and versatile building blocks for nanotechnology. Here we demonstrate the interaction of SeNPs with BSA macromolecule simply by physical adsorption method. The interaction between SeNPs and BSA has been investigated by UV-Vis, fluorescence, circular dichroism (CD) spectroscopic and thermal methods. The esterase-like activity of BSA towards PNPA was investigated in the presence of SeNPs. The effects of SeNPs on the stability and conformational changes of BSA were studied, which indicated that the binding of SeNPs with BSA induced relative changes in secondary structure of protein. SeNPs acted as a structure stabilizer for BSA which was further confirmed by thermal denaturation study. The hydrophilic bonding forces played important roles in the BSA-SeNPs complex formation. The putative binding site of SeNPs on BSA was near to Sudlow's site II. The hydrophilic interaction of SeNPs on the stability and structure of BSA would find promising application in drug delivery system.

Review on mechanisms and interactions in concomitant use of herbs and warfarin therapy.

The effectiveness of warfarin, an oral anticoagulant originally derived from a plant, is strongly affected by patient's characteristics such as the age, presence of comorbidities, and concomitant use of another drug. Warfarin has the potential to interact with many drugs, medicinal plants, and food, which increases the risk of adverse events. A critical analysis of scientific literature was conducted to assess the interferences of medicinal plants with blood haemostasis and then with warfarin anticoagulation. We found 58 different plants that may alter the blood haemostasis and anticoagulation with warfarin. The herbs that showed the greatest potential to interact with warfarin include garlic, ginger, ginkgo, St. John's wort, and ginseng, i.e. plants normally consumed as food and also used for therapeutic purposes. The interactions between drugs and herbs are varied because of the complex chemical matrix of plants. Mainly coumarins, quinones, xanthones, terpenes, lignans, and vitamin K showed significant influence on warfarin treatment. In general, these plants can potentiate the effect of warfarin by stimulating anticoagulation in multiple ways, and the clinical outcome associated with this interaction is the increase of bleeding risk. Moreover, potential interactions between herbal products and drugs are a safety concern, especially for drugs with a narrow therapeutic index or for patients receiving drug treatment for chronic diseases, and both of these apply to warfarin pharmacotherapy. Therefore, this review article summarises the data on the influence of medicinal plants on warfarin treatment and analyses this information in view of the interaction targets. The relevant plants were categorised according to their target, and their effects are discussed in order to organise the isolated information and to highlight the need of further discussion and new studies on the safety of herbal medicines and warfarin.

The effects of ferulic acid on the pharmacokinetics of warfarin in rats after biliary drainage.

According to previous research studies, warfarin can be detected in human bile after oral administration. Ferulic acid (FA) is the main bioactive component of many Chinese herbs for the treatment of cardiovascular disease. To elucidate the effects of FA on the pharmacokinetics of warfarin in rats after biliary drainage is necessary. Twenty rats were randomly divided into four groups: Group 1 (WN): healthy rats after the administration of warfarin sodium, Group 2 (WO): a rat model of biliary drainage after the administration of warfarin sodium, Group 3 (WFN): healthy rats after the administration of warfarin sodium and FA, and Group 4 (WFO): a rat model of biliary drainage after the administration of warfarin sodium and FA. Blood samples were collected at different time points after administration. The concentrations of blood samples were determined by ultraperformance liquid chromatography-tandem mass spectrometry. Comparisons between groups were performed according to the main pharmacokinetic parameters calculated by the DAS 2.1.1 software. The pharmacokinetic parameters showed a significant difference between the WN and WO groups, the WO group showed a decrease of 51% and 41.6% in area under the curve from 0 to time (AUC0- t ) and peak plasma concentration (C max), respectively, whereas time to C max (T max) was delayed 3.27 folds. There were significant differences between the WFO and WFN groups, the WFO group showed a decrease of 63.8% and 70% in AUC0- t and C max, respectively; the delay in T max between the WN and WFN groups (mean, from 132-432 minutes) was significantly different; the mean retention time from 0 to time (MRT0- t ) between the WO and WFO groups (mean, from 718.31-606.13 minutes) also showed a significant difference. Enterohepatic circulation markedly influences the disposition of warfarin in rats, and FA significantly affected the warfarin disposition in rat plasma.

Interaction of lafutidine in binding to human serum albumin in gastric ulcer therapy: STD-NMR, WaterLOGSY-NMR, NMR relaxation times, Tr-NOESY, molecule docking, and spectroscopic studies.

In this study, lafutidine (LAF) was used as a model compound to investigate the binding mechanism between antiulcer drugs and human serum albumin (HSA) through various techniques, including STD-NMR, WaterLOGSY-NMR, (1)H NMR relaxation times, tr-NOESY, molecule docking calculation, FT-IR spectroscopy, and CD spectroscopy. The analyses of STD-NMR, which derived relative STD (%) intensities, and WaterLOGSY-NMR, determined that LAF bound to HSA. In particular, the pyridyl group of LAF was in close contact with HSA binding pocket, whereas furyl group had a secondary binding. Competitive STD-NMR and WaterLOGSY-NMR experiments, with warifarin and ibuprofen as site-selective probes, indicated that LAF preferentially bound to site II in the hydrophobic subdomains IIIA of HSA. The bound conformation of LAF at the HSA binding site was further elucidated by transferred NOE effect (tr-NOESY) experiment. Relaxation experiments provided quantitative information about the relationship between the affinity and structure of LAF. The molecule docking simulations conducted with AutoDock and the restraints derived from STD results led to three-dimensional models that were consistent with the NMR spectroscopic data. The presence of hydrophobic forces and hydrogen interactions was also determined. Additionally, FT-IR and CD spectroscopies showed that LAF induced secondary structure changes of HSA.

Assessment of Binding Affinity between Drugs and Human Serum Albumin Using Nanoporous Anodic Alumina Photonic Crystals.

In this study, we report an innovative approach aiming to assess the binding affinity between drug molecules and human serum albumin by combining nanoporous anodic alumina rugate filters (NAA-RFs) modified with human serum albumin (HSA) and reflectometric interference spectroscopy (RIfS). NAA-RFs are photonic crystal structures produced by sinusoidal pulse anodization of aluminum that present two characteristic optical parameters, the characteristic reflection peak (λPeak), and the effective optical thickness of the film (OTeff), which can be readily used as sensing parameters. A design of experiments strategy and an ANOVA analysis are used to establish the effect of the anodization parameters (i.e., anodization period and anodization offset) on the sensitivity of HSA-modified NAA-RFs toward indomethacin, a model drug. To this end, two sensing parameters are used, that is, shifts in the characteristic reflection peak (ΔλPeak) and changes in the effective optical thickness of the film (ΔOTeff). Subsequently, optimized NAA-RFs are used as sensing platforms to determine the binding affinity between a set of drugs (i.e., indomethacin, coumarin, sulfadymethoxine, warfarin, and salicylic acid) and HSA molecules. Our results verify that the combination of HSA-modified NAA-RFs with RIfS can be used as a portable, low-cost, and simple system for establishing the binding affinity between drugs and plasma proteins, which is a critical factor to develop efficient medicines for treating a broad range of diseases and medical conditions.

Comparative inhibitory effect of prenylated coumarins, ferulenol and ferprenin, contained in the 'poisonous chemotype' of Ferula communis on mammal liver microsomal VKORC1 activity.

Two distinguishable chemotypes of Ferula communis have been described: the 'nonpoisonous' chemotype, containing as main constituents the daucane esters; and the 'poisonous' chemotype containing prenylated coumarins, such as ferulenol and ferprenin. Ferulenol and ferprenin are 4-oxygenated molecules such as dicoumarol and warfarin, the first developed antivitamin K molecules. Antivitamin K molecules specifically inhibit VKORC1, an enzyme essential for recycling vitamin K. This latest is involved in the activation of clotting factors II, VII, IX, X. The inhibiting effect of ferulenol on VKORC1 was shown in rat, but not for species exposed to F. communis while in vivo studies suggest differences between animal susceptibility to ferulenol. The inhibiting effect of ferprenin on VKORC1 was never demonstrated. The aim of this study was to compare the inhibiting effect of both compounds on VKORC1 of different species exposed to F. communis. Vitamin K epoxide activity was evaluated for each species from liver microsomes and inhibiting effect of ferulenol and ferprenin was characterized. Ferulenol and ferprenin were shown to be able to inhibit VKORC1 from all analyzed species. Nevertheless, susceptibility to ferulenol and ferprenin presented differences between species, suggesting a different susceptibility to 'poisonous' chemotypes of F. communis.

Protein-based fluorescent metal nanoclusters for small molecular drug screening.

A facile drug screening method based on synthesis of fluorescent gold nanoclusters inside albumin proteins loaded with small molecular drugs and comparing the relative fluorescence intensities of the resultant gold nanoclusters has been developed and successfully applied for the quantitative measurement of drug-protein binding constants.

Cys34-cysteinylated human serum albumin is a sensitive plasma marker in oxidative stress-related chronic diseases.

The degree of oxidized cysteine (Cys) 34 in human serum albumin (HSA), as determined by high performance liquid chromatography (HPLC), is correlated with oxidative stress related pathological conditions. In order to further characterize the oxidation of Cys34-HSA at the molecular level and to develop a suitable analytical method for a rapid and sensitive clinical laboratory analysis, the use of electrospray ionization time-of-flight mass spectrometer (ESI-TOFMS) was evaluated. A marked increase in the cysteinylation of Cys34 occurs in chronic liver and kidney diseases and diabetes mellitus. A significant positive correlation was observed between the Cys-Cys34-HSA fraction of plasma samples obtained from 229 patients, as determined by ESI-TOFMS, and the degree of oxidized Cys34-HSA determined by HPLC. The Cys-Cys34-HSA fraction was significantly increased with the progression of liver cirrhosis, and was reduced by branched chain amino acids (BCAA) treatment. The changes in the Cys-Cys34-HSA fraction were significantly correlated with the alternations of the plasma levels of advanced oxidized protein products, an oxidative stress marker for proteins. The binding ability of endogenous substances (bilirubin and tryptophan) and drugs (warfarin and diazepam) to HSA purified from chronic liver disease patients were significantly suppressed but significantly improved by BCAA supplementation. Interestingly, the changes in this physiological function of HSA in chronic liver disease were correlated with the Cys-Cys34-HSA fraction. In conclusion, ESI-TOFMS is a suitable high throughput method for the rapid and sensitive quantification of Cys-Cys34-HSA in a large number of samples for evaluating oxidative stress related chronic disease progression or in response to a treatment.

A cellular system for quantitation of vitamin K cycle activity: structure-activity effects on vitamin K antagonism by warfarin metabolites.

Warfarin and other 4-hydroxycoumarins inhibit vitamin K epoxide reductase (VKOR) by depleting reduced vitamin K that is required for posttranslational modification of vitamin K-dependent clotting factors. In vitro prediction of the in vivo potency of vitamin K antagonists is complicated by the complex multicomponent nature of the vitamin K cycle. Here we describe a sensitive assay that enables quantitative analysis of γ-glutamyl carboxylation and its antagonism in live cells. We engineered a human embryonic kidney (HEK) 293-derived cell line (HEK 293-C3) to express a chimeric protein (F9CH) comprising the Gla domain of factor IX fused to the transmembrane and cytoplasmic regions of proline-rich Gla protein 2. Maximal γ-glutamyl carboxylation of F9CH required vitamin K supplementation, and was dose-dependently inhibited by racemic warfarin at a physiologically relevant concentration. Cellular γ-glutamyl carboxylation also exhibited differential VKOR inhibition by warfarin enantiomers (S > R) consistent with their in vivo potencies. We further analyzed the structure-activity relationship for inhibition of γ-glutamyl carboxylation by warfarin metabolites, observing tolerance to phenolic substitution at the C-5 and especially C-6, but not C-7 or C-8, positions on the 4-hydroxycoumarin nucleus. After correction for in vivo concentration and protein binding, 10-hydroxywarfarin and warfarin alcohols were predicted to be the most potent inhibitory metabolites in vivo.

Structure and stability of warfarin-sodium inclusion complexes formed with permethylated monoamino-ß-cyclodextrin.

Inclusion complexes of warfarin enantiomers with permethylated monoamino-ß-cyclodextrin (PMMABCD) were characterized using CE and (1)H NMR spectroscopy in aqueous solution. These techniques gave complementary information on the stability and the structure of the diastereomeric host-guest inclusion complexes. The stability constants were determined from CE experiments in a wide pH range. Change in the migration order on the variation of the pH was observed. (1)H NMR assignments have been established for the seven non-equivalent carbohydrate units of the host in the complex at pH 7-9. Specific H-H distance restraints were obtained from NOESY experiments and were introduced into molecular modeling to establish the geometry of the inclusion complexes. It was found that the open side chain warfarin enters the cavity from the primary side of the CD. The orientation of the coumarin ring within the cavity has the same preference for the two warfarin enantiomers owing to an ionic interaction with the amino group of the CD. Accordingly, enantioselectivity at pH 8.5 arises from the difference in the CH/π interactions between warfarin aromatics and the manifold of CH groups of the CD.

Two flavonolignans from milk thistle (Silybum marianum) inhibit CYP2C9-mediated warfarin metabolism at clinically achievable concentrations.

Milk thistle (Silybum marianum) is a popular herbal product used for hepatoprotection and chemoprevention. Two commercially available formulations are the crude extract, silymarin, and the semipurified product, silibinin. Silymarin consists of at least seven flavonolignans, of which the most prevalent are the diastereoisomers silybin A and silybin B; silibinin consists only of silybin A and silybin B. Based on a recent clinical study showing an interaction between a silymarin product and the CYP2C9 substrate losartan, the CYP2C9 inhibition properties of silybin A and silybin B and corresponding regioisomers, isosilybin A and isosilybin B, were evaluated using human liver microsomes (HLMs), recombinant CYP2C9 (rCYP2C9) enzymes, and the clinically relevant probe, (S)-warfarin. Silybin B was the most potent inhibitor in HLMs, followed by silybin A, isosilybin B, and isosilybin A (IC(50) of 8.2, 18, 74, and >100 microM, respectively). Next, silybin A and silybin B were selected for further characterization. As with HLMs, silybin B was more potent than silybin A toward rCYP2C9 1 (6.7 versus 12 microM), rCYP2C9 2 (9.3 versus 19 microM), and rCYP2C9 3 (2.4 versus 9.3 microM). Using a matrix of five substrate (1-15 microM) and six inhibitor (1-80 microM) concentrations and HLMs, both diastereoisomers inhibited (S)-warfarin 7-hydroxylation in a manner described best by a mixed-type inhibition model (K(i) values of 4.8 and 10 microM for silybin B and silybin A, respectively). These observations, combined with the high systemic silibinin concentrations (>5-75 microM) achieved in a phase I study involving prostate cancer patients, prompt clinical evaluation of a potential warfarin-milk thistle interaction.

Combined approach for high-throughput preparation and analysis of plasma samples from exposure studies.

In drug discovery today, drug exposure is determined in preclinical efficacy and safety studies and drug effects are related to measured concentrations rather than to the administered dose. This leads to a strong increase in the number of bioanalytical samples, demanding the development of higher throughput methods to cope with the increased workload. Here, a combined approach is described for the high-throughput preparation and liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of drug levels in plasma samples from the preclinical efficacy and safety studies, i.e. exposure studies. Appropriate pharmacokinetic (PK) compartmental models were fitted to data from PK screening studies in the rat, which were subsequently used to simulate the expected plasma concentrations of the respective exposure studies. Information on the estimated drug concentrations was used to dilute the samples to appropriate concentration levels. A Tecan Genesis RSP liquid handling system was utilized to perform automated plasma sample preparation including serial dilution of standard solutions, dilution of plasma samples, addition of internal standard solution and precipitation with acetonitrile. This robotic sample preparation process permitted two studies of 1-96 samples each to be run simultaneously. To ensure the performance of this method the accuracy and precision for diazepam were examined. Two novel drugs were used to illustrate the suggested approach. In conclusion, our method for sample preparation of exposure samples, based on the combined use of PK simulations, a liquid handling system and a fast LC/MS/MS method, increased the throughput more than three times and minimized the errors, while maintaining the required accuracy and precision.

Plasma S/R ratio of warfarin co-varies with VKORC1 haplotype.

We recently reported that the low-dose VKORC1*2 haplotype is an important genetic determinant for warfarin dose requirement and is associated with difficulties to attain stable therapeutic prothrombin time--International Normalized Ratio in patients undergoing anticoagulation therapy. The aim of this study was to investigate whether patients with VKORC1*2 compared with patients carrying high-dose haplotypes VKORC1*3 or VKORC1*4 had different warfarin S/R ratios in their plasma, and whether that was related to CYP2C9 variants CYP2C9*2 and CYP2C9*3 or other factors. Samples from patients previously haplotyped for VKORC1 and measured for plasma warfarin concentration were genotyped for the CYP2C9 variants CYP2C9*2 and CYP2C9*3. Nonparametric statistical analysis was performed to elucidate whether there was any significant difference in the warfarin S/R ratio between the two patient groups. Our result shows that there is a significant difference (P<0.01) in warfarin S/R ratios between VKORC1*2 and VKORC1*3 or VKORC1*4 patients. This difference did not originate from CYP2C9 variants CYP2C9*2 and CYP2C9*3. We speculate that VKORC1 haplotypes possibly are linked to some unidentified factors involved in the metabolic clearance of warfarin enantiomers. Dose-dependent variations in (S)-warfarin and (R)-warfarin clearance in these patients can also be a probable explanation for the difference in warfarin S/R ratios.

Interaction of warfarin with drugs, natural substances, and foods.

Variability in the anticoagulant response to warfarin is an ongoing clinical dilemma. Fluctuations in dietary vitamin K are an important source of variance, and the need for constancy in vitamin K intake is routinely emphasized for warfarin-treated patients. Anticoagulant response is also influenced by a number of drugs that induce or inhibit warfarin metabolism, as well as by genetic polymorphisms that may modulate expression or activity of CYP2C9, the isoform mediating clearance of S-warfarin. The possible role of dietary factors other than vitamin K, as well as of herbal medicines or supplements as contributors to the instability of anticoagulation in warfarin-treated patients, has received recent attention. St. John's wort and possibly some ginseng formulations may have the potential to diminish warfarin anticoagulation, apparently by inducing CYP2C9 activity. Otherwise, there is no reliable evidence to indicate that any dietary component (other than vitamin K) or any herbal product has an effect on the anticoagulant response to warfarin. Scientific conclusions on this important therapeutic issue should be based on valid scientific data rather than unvalidated case reports.

Evaluation of a micro volume pulsed ultrafiltration cell for screening ligands in non-covalent complexes.

A pulsed ultrafiltration cell with a 35 microL binding chamber was evaluated for its ability to screen ligands that formed non-covalent complexes with protein targets. The cell was tested with ligands to the targets of carbonic anhydrase and serum albumin. Non-covalent ligand binding to both of these targets was observed and bound ligands were eluted from the cell in less than five min. The cell was also demonstrated to effectively screen a methanolic fermentation broth extract spiked with a known inhibitor to carbonic anhydrase. In addition to detecting specific binding events, the pulsed ultrafiltration method was investigated for its ability to distinguish non-specific binding events. Using carbonic anhydrase with the zinc-binding site removed, it was found that non-specific complexes observed when using electrospray ionization alone were not detected when using the pulsed ultrafiltration mass spectrometry method.