Andexanet for Factor Xa Inhibitor-Associated Acute Intracerebral Hemorrhage.

BACKGROUND: Patients with acute intracerebral hemorrhage who are receiving factor Xa inhibitors have a risk of hematoma expansion. The effect of andexanet alfa, an agent that reverses the effects of factor Xa inhibitors, on hematoma volume expansion has not been well studied. METHODS: We randomly assigned, in a 1:1 ratio, patients who had taken factor Xa inhibitors within 15 hours before having an acute intracerebral hemorrhage to receive andexanet or usual care. The primary end point was hemostatic efficacy, defined by expansion of the hematoma volume by 35% or less at 12 hours after baseline, an increase in the score on the National Institutes of Health Stroke Scale of less than 7 points (scores range from 0 to 42, with higher scores indicating worse neurologic deficit) at 12 hours, and no receipt of rescue therapy between 3 hours and 12 hours. Safety end points were thrombotic events and death. RESULTS: A total of 263 patients were assigned to receive andexanet, and 267 to receive usual care. Efficacy was assessed in an interim analysis that included 452 patients, and safety was analyzed in all 530 enrolled patients. Atrial fibrillation was the most common indication for factor Xa inhibitors. Of the patients receiving usual care, 85.5% received prothrombin complex concentrate. Hemostatic efficacy was achieved in 150 of 224 patients (67.0%) receiving andexanet and in 121 of 228 (53.1%) receiving usual care (adjusted difference, 13.4 percentage points; 95% confidence interval [CI], 4.6 to 22.2; P = 0.003). The median reduction from baseline to the 1-to-2-hour nadir in anti-factor Xa activity was 94.5% with andexanet and 26.9% with usual care (P<0.001). Thrombotic events occurred in 27 of 263 patients (10.3%) receiving andexanet and in 15 of 267 (5.6%) receiving usual care (difference, 4.6 percentage points; 95% CI, 0.1 to 9.2; P = 0.048); ischemic stroke occurred in 17 patients (6.5%) and 4 patients (1.5%), respectively. There were no appreciable differences between the groups in the score on the modified Rankin scale or in death within 30 days. CONCLUSIONS: Among patients with intracerebral hemorrhage who were receiving factor Xa inhibitors, andexanet resulted in better control of hematoma expansion than usual care but was associated with thrombotic events, including ischemic stroke. (Funded by Alexion AstraZeneca Rare Disease and others; ANNEXA-I ClinicalTrials.gov number, NCT03661528.).

Association between heavy alcohol consumption and cryptogenic ischaemic stroke in young adults: a case-control study.

BACKGROUND: The underlying risk factors for young-onset cryptogenic ischaemic stroke (CIS) remain unclear. This multicentre study aimed to explore the association between heavy alcohol consumption and CIS with subgroup analyses stratified by sex and age. METHODS: Altogether, 540 patients aged 18-49 years (median age 41; 47.2% women) with a recent CIS and 540 sex-matched and age-matched stroke-free controls were included. Heavy alcohol consumption was defined as >7 (women) and >14 (men) units per week or at least an average of two times per month ≥5 (women) and ≥7 (men) units per instance (binge drinking). A conditional logistic regression adjusting for age, sex, education, hypertension, cardiovascular diseases, diabetes, hypercholesterolaemia, current smoking, obesity, diet and physical inactivity was used to assess the independent association between alcohol consumption and CIS. RESULTS: Patients were twice as more often heavy alcohol users compared with controls (13.7% vs 6.7%, p<0.001), were more likely to have hypertension and they were more often current smokers, overweight and physically inactive. In the entire study population, heavy alcohol consumption was independently associated with CIS (adjusted OR 2.11; 95% CI 1.22 to 3.63). In sex-specific analysis, heavy alcohol consumption was associated with CIS in men (2.72; 95% CI 1.25 to 5.92), but not in women (1.56; 95% CI 0.71 to 3.41). When exploring the association with binge drinking alone, a significant association was shown in the entire cohort (2.43; 95% CI 1.31 to 4.53) and in men (3.36; 95% CI 1.44 to 7.84), but not in women. CONCLUSIONS: Heavy alcohol consumption, particularly binge drinking, appears to be an independent risk factor in young men with CIS.

Neuroprotective Effects of Inhaled Xenon Gas on Brain Structural Gray Matter Changes After Out-of-Hospital Cardiac Arrest Evaluated by Morphometric Analysis: A Substudy of the Randomized Xe-Hypotheca Trial.

BACKGROUND: We have earlier reported that inhaled xenon combined with hypothermia attenuates brain white matter injury in comatose survivors of out-of-hospital cardiac arrest (OHCA). A predefined secondary objective was to assess the effect of inhaled xenon on the structural changes in gray matter in comatose survivors after OHCA. METHODS: Patients were randomly assigned to receive either inhaled xenon combined with target temperature management (33 °C) for 24 h (n = 55, xenon group) or target temperature management alone (n = 55, control group). A change of brain gray matter volume was assessed with a voxel-based morphometry evaluation of high-resolution structural brain magnetic resonance imaging (MRI) data with Statistical Parametric Mapping. Patients were scheduled to undergo the first MRI between 36 and 52 h and a second MRI 10 days after OHCA. RESULTS: Of the 110 randomly assigned patients in the Xe-Hypotheca trial, 66 patients completed both MRI scans. After all imaging-based exclusions, 21 patients in the control group and 24 patients in the xenon group had both scan 1 and scan 2 available for analyses with scans that fulfilled the quality criteria. Compared with the xenon group, the control group had a significant decrease in brain gray matter volume in several clusters in the second scan compared with the first. In a between-group analysis, significant reductions were found in the right amygdala/entorhinal cortex (p = 0.025), left amygdala (p = 0.043), left middle temporal gyrus (p = 0.042), left inferior temporal gyrus (p = 0.008), left parahippocampal gyrus (p = 0.042), left temporal pole (p = 0.042), and left cerebellar cortex (p = 0.005). In the remaining gray matter areas, there were no significant changes between the groups. CONCLUSIONS: In comatose survivors of OHCA, inhaled xenon combined with targeted temperature management preserved gray matter better than hypothermia alone. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov: NCT00879892.

Comparison of the prognostic value of early-phase proton magnetic resonance spectroscopy and diffusion tensor imaging with serum neuron-specific enolase at 72 h in comatose survivors of out-of-hospital cardiac arrest-a substudy of the XeHypotheca trial.

PURPOSE: We compared the predictive accuracy of early-phase brain diffusion tensor imaging (DTI), proton magnetic resonance spectroscopy (1H-MRS), and serum neuron-specific enolase (NSE) against the motor score and epileptic seizures (ES) for poor neurological outcome after out-of-hospital cardiac arrest (OHCA). METHODS: The predictive accuracy of DTI, 1H-MRS, and NSE along with motor score at 72 h and ES for the poor neurological outcome (modified Rankin Scale, mRS, 3 - 6) in 92 comatose OHCA patients at 6 months was assessed by area under the receiver operating characteristic curve (AUROC). Combined models of the variables were included as exploratory. RESULTS: The predictive accuracy of fractional anisotropy (FA) of DTI (AUROC 0.73, 95% CI 0.62-0.84), total N-acetyl aspartate/total creatine (tNAA/tCr) of 1H-MRS (0.78 (0.68 - 0.88)), or NSE at 72 h (0.85 (0.76 - 0.93)) was not significantly better than motor score at 72 h (0.88 (95% CI 0.80-0.96)). The addition of FA and tNAA/tCr to a combination of NSE, motor score, and ES provided a small but statistically significant improvement in predictive accuracy (AUROC 0.92 (0.85-0.98) vs 0.98 (0.96-1.00), p = 0.037). CONCLUSION: None of the variables (FA, tNAA/tCr, ES, NSE at 72 h, and motor score at 72 h) differed significantly in predicting poor outcomes in this patient group. Early-phase quantitative neuroimaging provided a statistically significant improvement for the predictive value when combined with ES and motor score with or without NSE. However, in clinical practice, the additional value is small, and considering the costs and challenges of imaging in this patient group, early-phase DTI/MRS cannot be recommended for routine use. TRIAL REGISTRATION: ClinicalTrials.gov NCT00879892, April 13, 2009.

Atrial fibrillation after closure of patent foramen ovale in the REDUCE clinical study.

OBJECTIVES: To describe the occurrence of postprocedural atrial fibrillation (AF) among patients with cryptogenic stroke undergoing patent foramen ovale (PFO) closure in the REDUCE clinical study and analyze for potential risk factors for the development of postprocedural AF. BACKGROUND: AF is an adverse event that might potentially counterbalance the stroke prevention benefit from PFO closure. Data on AF after transcatheter PFO closure are sparse. METHODS: We evaluated data from patients having PFO closure (Gore HELEX or Gore Cardioform Septal Occluder) in the REDUCE clinical trial (n = 408) in at post hoc explorative analysis. Median follow-up was 5.0 years. RESULTS: AF occurred in 30 patients (7.4%) after PFO closure with a total of 34 AF events. Most were reported as non-serious (68%), detected within 45 days post-procedure (79%), and resolved within 2 weeks of onset (63%). One subject with AF had recurrent stroke. Postprocedural AF occurred more frequently among subjects with higher age and large device sizes. Male sex was the only independent predictor of postprocedural AF. We found no association between the type of occluder (HELEX or Gore Cardioform Septal Occluder) or PFO anatomical characteristics and post-procedural AF. CONCLUSION: In the REDUCE clinical study, postprocedural atrial fibrillation was mostly early onset, transient and with no later recurrence. Postprocedural AF occurred more frequently among patients with higher age and larger devices. Male sex was the only independent predictor of postprocedural AF.

In vitro metabolism of helenalin and its inhibitory effect on human cytochrome P450 activity.

Sesquiterpene lactone helenalin is used as an antiphlogistic in European and Chinese folk medicine. The pharmacological activities of helenalin have been extensively investigated, yet insufficient information exists about its metabolic properties. The objectives of the present study were (1) to investigate the in vitro NADPH-dependent metabolism of helenalin (5 and 100 µM) using human and rat liver microsomes and liver cytosol, (2) to elucidate the role of human cytochrome P450 (CYP) enzymes in its oxidative metabolism, and (3) to study the inhibition of human CYPs by helenalin. Five oxidative metabolites were detected in NADPH-dependent human and rat liver microsomal incubations, while two reduced metabolites were detected only in NADPH-dependent human microsomal and cytosolic incubations. In human liver microsomes, the main oxidative metabolite was 14-hydroxyhelenalin, and in rat liver microsomes 9-hydroxyhelenalin. The overall oxidation of helenalin was several times more efficient in rat than in human liver microsomes. In humans, CYP3A4 and CYP3A5 followed by CYP2B6 were the main enzymes responsible for the hepatic metabolism of helenalin. The extrahepatic CYP2A13 oxidized helenalin most efficiently among CYP enzymes, possessing the Km value of 0.6 µM. Helenalin inhibited CYP3A4 (IC50 = 18.7 µM) and CYP3A5 (IC50 = 62.6 µM), and acted as a mechanism-based inhibitor of CYP2A13 (IC50 = 1.1 µM, KI = 6.7 µM, and kinact = 0.58 ln(%)/min). It may be concluded that the metabolism of helenalin differs between rats and humans, in the latter its oxidation is catalyzed by hepatic CYP2B6, CYP3A4, CYP3A5, and CYP3A7, and extrahepatic CYP2A13.

Obesity and the Risk of Cryptogenic Ischemic Stroke in Young Adults.

OBJECTIVES: We examined the association between obesity and early-onset cryptogenic ischemic stroke (CIS) and whether fat distribution or sex altered this association. MATERIALS AND METHODS: This prospective, multi-center, case-control study included 345 patients, aged 18-49 years, with first-ever, acute CIS. The control group included 345 age- and sex-matched stroke-free individuals. We measured height, weight, waist circumference, and hip circumference. Obesity metrics analyzed included body mass index (BMI), waist-to-hip ratio (WHR), waist-to-stature ratio (WSR), and a body shape index (ABSI). Models were adjusted for age, level of education, vascular risk factors, and migraine with aura. RESULTS: After adjusting for demographics, vascular risk factors, and migraine with aura, the highest tertile of WHR was associated with CIS (OR for highest versus lowest WHR tertile 2.81, 95%CI 1.43-5.51; P=0.003). In sex-specific analyses, WHR tertiles were not associated with CIS. However, using WHO WHR cutoff values (>0.85 for women, >0.90 for men), abdominally obese women were at increased risk of CIS (OR 2.09, 95%CI 1.02-4.27; P=0.045). After adjusting for confounders, WC, BMI, WSR, or ABSI were not associated with CIS. CONCLUSIONS: Abdominal obesity measured with WHR was an independent risk factor for CIS in young adults after rigorous adjustment for concomitant risk factors.

Patent Foramen Ovale Closure Decreases the Incidence but Not the Size of New Brain Infarction on Magnetic Resonance Imaging: An Analysis of the REDUCE Trial.

Background and Purpose: Randomized patent foramen ovale closure trials have used open-label end point ascertainment which increases the risk of bias and undermines confidence in the conclusions. The Gore REDUCE trial prospectively performed baseline and follow-up magnetic resonance imaging (MRIs) for all subjects providing an objective measure of the effectiveness of closure. Methods: We performed blinded evaluations of the presence, location, and volume of new infarct on diffusion-weighted imaging of recurrent clinical stroke or new infarct (>3 mm) on T2/fluid attenuated inversion recovery from baseline to follow-up MRI at 2 years, comparing closure to medical therapy alone. We also examined the effect of shunt size and the development of atrial fibrillation on infarct burden at follow-up. Results: At follow-up, new clinical stroke or silent MRI infarct occurred in 18/383 (4.7%) patients who underwent closure and 19/177 (10.7%) medication-only patients (relative risk, 0.44 [95% CI, 0.24­0.81], P=0.02). Clinical strokes were less common in closure patients compared with medically treated patients, 5 (1.3%) versus 12 (6.8%), P=0.001, while silent MRI infarcts were similar, 13 (3.4%) versus 7 (4.0%), P=0.81. There were no differences in number, volumes, and distribution of new infarct comparing closure patients to those treated with medication alone. There were also no differences of number, volumes, and distribution comparing silent infarcts to clinical strokes. Infarct burden was also similar for patients who developed atrial fibrillation and for those with large shunts. Conclusions: The REDUCE trial demonstrates that patent foramen ovale closure prevents recurrent brain infarction based on the objective outcome of new infarcts on MRI. Only clinical strokes were reduced by closure while silent infarctions were similar between study arms, and there were no differences in infarct volume or location comparing silent infarcts to clinical strokes. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT00738894.

Tissue and interspecies comparison of catechol-O-methyltransferase mediated catalysis of 6-O-methylation of esculetin to scopoletin and its inhibition by entacapone and tolcapone.

Catechol-O-methyltransferase (COMT) methylates both endogenous and exogenous catechol compounds to inactive and safe metabolites. We first optimised conditions for a convenient and sensitive continuous fluorescence-based 6-O-methylation assay of esculetin, which we used for investigating the COMT activity in human, mouse, rat, dog, rabbit, and sheep liver cytosols and microsomes and in ten different rat tissues. Furthermore, we compared the inhibition potencies and mechanisms of two clinically used COMT inhibitors, entacapone and tolcapone, in these species. In most tissues, the COMT activity was at least three times higher in cytosol than in microsomes. In the rat, the highest COMT activity was found in the liver, followed by kidney, ileum, thymus, spleen, lung, pancreas, heart, brain, and finally, skeletal muscle. Entacapone and tolcapone were characterised as highly potent mixed type tight-binding inhibitors. The competitive inhibition type dominated over the uncompetitive inhibition with entacapone, whereas uncompetitive inhibition dominated with tolcapone. Rats, dogs, pigs, and sheep are high COMT activity species, in contrast to humans, mice, and rabbits; COMT activity is highest in the liver. Both entacapone and tolcapone are potent COMT inhibitors, but their inhibition mechanisms differ.

Molecular docking and oxidation kinetics of 3-phenyl coumarin derivatives by human CYP2A13.

CYP2A13 enzyme is expressed in human extrahepatic tissues, while CYP2A6 is a hepatic enzyme. Reactions catalysed by CYP2A13 activate tobacco-specific nitrosamines and some other toxic xenobiotics in lungs.To compare oxidation characteristics and substrate-enzyme active site interactions in CYP2A13 vs CYP2A6, we evaluated CYP2A13 mediated oxidation characteristics of 23 coumarin derivatives and modelled their interactions at the enzyme active site.CYP2A13 did not oxidise six coumarin derivatives to corresponding fluorescent 7-hydroxycoumarins. The Km-values of the other coumarins varied 0.85-97 µM, Vmax-values of the oxidation reaction varied 0.25-60 min-1, and intrinsic clearance varied 26-6190 kL/min*mol CYP2A13). Km of 6-chloro-3-(3-hydroxyphenyl)-coumarin was 0.85 (0.55-1.15 95% confidence limit) µM and Vmax 0.25 (0.23-0.26) min-1, whereas Km of 6-hydroxy-3-(3-hydroxyphenyl)-coumarin was 10.9 (9.9-11.8) µM and Vmax 60 (58-63) min-1. Docking analyses demonstrated that 6-chloro or 6-methoxy and 3-(3-hydroxyphenyl) or 3-(4-trifluoromethylphenyl) substituents of coumarin increased affinity to CYP2A13, whereas 3-triazole or 3-(3-acetate phenyl) or 3-(4-acetate phenyl) substituents decreased it.The active site of CYP2A13 accepts more diversified types of coumarin substrates than the hepatic CYP2A6 enzyme. New sensitive and convenient profluorescent CYP2A13 substrates were identified, such as 6-chloro-3-(3-hydroxyphenyl)-coumarin having high affinity and 6-hydroxy-3-(3-hydroxyphenyl)-coumarin with high intrinsic clearance.

Atrial Cardiopathy and Nonstenosing Large Artery Plaque in Patients With Embolic Stroke of Undetermined Source.

Background and Purpose- Atrial cardiopathy and atherosclerotic plaque are two potential mechanisms underlying embolic strokes of undetermined source (ESUS). The relationship between these two mechanisms among ESUS patients remains unclear. A better understanding of their association may inform targeted secondary prevention strategies. Methods- We examined the association between atrial cardiopathy and atherosclerotic plaque in the NAVIGATE ESUS trial (New Approach Rivaroxaban Inhibition of Factor Xa in a Global Trial Versus ASA to Prevent Embolism in Embolic Stroke of Undetermined Source), which enrolled 7213 patients with recent ESUS during 2014 to 2017. For this analysis, we included patients with data on left atrial dimension, location of brain infarction, and cervical large artery plaque. The variables of primary interest were left atrial diameter and cervical plaque ipsilateral to brain infarction. Secondary markers of atrial cardiopathy were premature atrial contractions on Holter monitoring and newly diagnosed atrial fibrillation. For descriptive purposes, left atrial enlargement was defined as ≥4.7 cm. Multivariable logistic regression was used to examine the association between atrial cardiopathy markers and ipsilateral plaque after adjustment for age, sex, body mass index, hypertension, diabetes mellitus, current smoking, and hyperlipidemia. Results- Among 3983 eligible patients, 235 (5.9%) had left atrial enlargement, 939 (23.6%) had ipsilateral plaque, and 94 (2.4%) had both. Shared risk factors for left atrial enlargement and ipsilateral plaque were male sex, white race, hypertension, tobacco use, and coronary artery disease. Despite shared risk factors, increasing left atrial dimension was not associated with ipsilateral plaque after adjustment for covariates (odds ratio per cm, 1.1 [95% CI, 1.0-1.2]; P=0.08). We found no consistent associations between secondary markers of atrial cardiopathy and ipsilateral plaque. Conclusions- In a large population of patients with ESUS, we did not observe a notable association between atrial cardiopathy and atherosclerotic plaque, and few patients had both conditions. These findings suggest that atrial cardiopathy and atherosclerotic plaque may be distinct, nonoverlapping risk factors for stroke among ESUS patients.

Patent Foramen Ovale Closure or Antiplatelet Therapy for Cryptogenic Stroke.

BACKGROUND: The efficacy of closure of a patent foramen ovale (PFO) in the prevention of recurrent stroke after cryptogenic stroke is uncertain. We investigated the effect of PFO closure combined with antiplatelet therapy versus antiplatelet therapy alone on the risks of recurrent stroke and new brain infarctions. METHODS: In this multinational trial involving patients with a PFO who had had a cryptogenic stroke, we randomly assigned patients, in a 2:1 ratio, to undergo PFO closure plus antiplatelet therapy (PFO closure group) or to receive antiplatelet therapy alone (antiplatelet-only group). Imaging of the brain was performed at the baseline screening and at 24 months. The coprimary end points were freedom from clinical evidence of ischemic stroke (reported here as the percentage of patients who had a recurrence of stroke) through at least 24 months after randomization and the 24-month incidence of new brain infarction, which was a composite of clinical ischemic stroke or silent brain infarction detected on imaging. RESULTS: We enrolled 664 patients (mean age, 45.2 years), of whom 81% had moderate or large interatrial shunts. During a median follow-up of 3.2 years, clinical ischemic stroke occurred in 6 of 441 patients (1.4%) in the PFO closure group and in 12 of 223 patients (5.4%) in the antiplatelet-only group (hazard ratio, 0.23; 95% confidence interval [CI], 0.09 to 0.62; P=0.002). The incidence of new brain infarctions was significantly lower in the PFO closure group than in the antiplatelet-only group (22 patients [5.7%] vs. 20 patients [11.3%]; relative risk, 0.51; 95% CI, 0.29 to 0.91; P=0.04), but the incidence of silent brain infarction did not differ significantly between the study groups (P=0.97). Serious adverse events occurred in 23.1% of the patients in the PFO closure group and in 27.8% of the patients in the antiplatelet-only group (P=0.22). Serious device-related adverse events occurred in 6 patients (1.4%) in the PFO closure group, and atrial fibrillation occurred in 29 patients (6.6%) after PFO closure. CONCLUSIONS: Among patients with a PFO who had had a cryptogenic stroke, the risk of subsequent ischemic stroke was lower among those assigned to PFO closure combined with antiplatelet therapy than among those assigned to antiplatelet therapy alone; however, PFO closure was associated with higher rates of device complications and atrial fibrillation. (Funded by W.L. Gore and Associates; Gore REDUCE ClinicalTrials.gov number, NCT00738894 .).

In vitro sulfonation of 7-hydroxycoumarin derivatives in liver cytosol of human and six animal species.

Sulfonation is an important high affinity elimination pathway for phenolic compounds.In this study sulfonation of 7-hydroxycoumarin and 13 its derivatives were evaluated in liver cytosols of human and six animal species. 7-hydroxycoumarin and its derivatives are strongly fluorescent, and their sulfate conjugates are nonfluorescent at excitation 405 nm and emission 460 nm. A convenient fluorescence based kinetic assay of sulfonation was established.The sulfonation rate of most of the 7-hydroxycoumarin derivatives was low in liver cytosol of human and pig, whereas it was high with most compounds in dog and intermediate in rat, mouse, rabbit, and sheep. Sulfonation of the 7-hydroxycoumarin derivatives followed Michaelis-Menten kinetics with Km values of 0.1-12 µM, Vmax of 0.005-1.7 µmol/(min * g protein) and intrinsic clearance (Vmax/Km) of 0.004-1.9 L/(min * g cytosolic protein).Fluorescence based measurement of sulfonation of 7-hydroxycoumarin derivatives provides a sensitive and convenient high-throughput assay to determine sulfonation rate in different species and tissues and can be applied to evaluate sulfonation kinetics and inhibition.

Coumarin-Based Profluorescent and Fluorescent Substrates for Determining Xenobiotic-Metabolizing Enzyme Activities In Vitro.

in vivo methods, such as spectrophotometric, fluorometric, mass spectrometric,and radioactivity-based techniques. In fluorescence-based assays, the reaction produces a fluorescentproduct from a nonfluorescent substrate or vice versa. Fluorescence-based enzyme assays areusually highly sensitive and specific, allowing measurements on small specimens of tissues withlow enzyme activities. Fluorescence assays are also amenable to miniaturization of the reactionmixtures and can thus be done in high throughput. 7-Hydroxycoumarin and its derivatives arewidely used as fluorophores due to their desirable photophysical properties. They possess a large -conjugated system with electron-rich and charge transfer properties. This conjugated structure leadsto applications of 7-hydroxycoumarins as fluorescent sensors for biological activities. We describe inthis review historical highlights and current use of coumarins and their derivatives in evaluatingactivities of the major types of xenobiotic-metabolizing enzyme systems. Traditionally, coumarinsubstrates have been used to measure oxidative activities of cytochrome P450 (CYP) enzymes. For thispurpose, profluorescent coumarins are very sensitive, but generally lack selectivity for individual CYPforms. With the aid of molecular modeling, we have recently described several new coumarin-basedsubstrates for measuring activities of CYP and conjugating enzymes with improved selectivity.

Comparison of Antiplatelet Therapies for Prevention of Patent Foramen Ovale-Associated Stroke.

AIMS: The REDUCE study demonstrated a reduction in the risk of recurrent stroke with patent foramen ovale closure and antiplatelet therapy compared to antiplatelet therapy alone. The clinicians were allowed to choose among aspirin, clopidogrel, or aspirin/dipyridamole with the expectation that all antiplatelet therapies would have similar efficacy in this population. We tested that presumption by comparing recurrent stroke rates among antiplatelet agents within the control arm of the trial. METHODS: We evaluated patients in REDUCE study who were randomized to the medical arm. The primary endpoint for this analysis was freedom from clinical ischemic stroke through at least 2 years of follow-up, to a maximum of 5 years. In the primary analysis, antiplatelet treatment was defined as the agent during the week prior to a recurrent stroke or last known contact. RESULTS: Of 223 patients in the medical treatment arm, the initial agent was aspirin 52%, clopidogrel 30%, and aspirin/dipyridamole 12%. Patients treated with aspirin were similar to those treated with alternatives, but were more likely to be enrolled in the United States. The last reported agent was aspirin alone in 55%, clopidogrel alone in 31%, aspirin/dipyridamole in 7%, and other/nothing/missing in 7%. Recurrent stroke rates were similar for all 3 antiplatelet regimens in unadjusted and adjusted analyses, with no overall difference among agents (P= .17). CONCLUSIONS: Among patients with patent foramen ovale-associated stroke who were managed medically, there were no differences among antiplatelet agents in the risk of recurrent stroke, though confidence intervals were wide.

Development of new Coumarin-based profluorescent substrates for human cytochrome P450 enzymes.

Cytochrome P450 (CYP) enzymes constitute an essential xenobiotic metabolizing system that regulates the elimination of lipophilic compounds from the body. Convenient and affordable assays for CYP enzymes are important for assessing these metabolic pathways. In this study, 10 novel profluorescent coumarin derivatives with various substitutions at carbons 3, 6 and 7 were developed. Molecular modeling indicated that 3-phenylcoumarin offers an excellent scaffold for the development of selective substrate compounds for various human CYP forms, as they could be metabolized to fluorescent 7-hydroxycoumarin derivatives. Oxidation of profluorescent coumarin derivatives to fluorescent metabolites by 13 important human liver xenobiotic-metabolizing CYP forms was determined by enzyme kinetic assays. Four of the coumarin derivatives were converted to fluorescent metabolites by CYP1 family enzymes, with 6-methoxy-3-(4-trifluoromethylphenyl)coumarin being oxidized selectively by CYP1A2 in human liver microsomes. Another set of four compounds were metabolized by CYP2A6 and CYP1 enzymes. 7-Methoxy-3-(3-methoxyphenyl)coumarin was oxidized efficiently by CYP2C19 and CYP2D6 in a non-selective fashion. The advantages of the novel substrates were (1) an excellent signal-to-background ratio, (2) selectivity for CYP1 forms, and (3) convenient multiwell plate measurement, allowing for precise determination of potential inhibitors of important human hepatic forms CYP1A2, CYP2C19 and CYP2D6.

Metabolism of Scoparone in Experimental Animals and Humans.

Scoparone, a major constituent of the Chinese herbal medicine Yin Chen Hao, expresses beneficial effects in experimental models of various diseases. The intrinsic doses and effects of scoparone are dependent on its metabolism, both in humans and animals. We evaluated in detail the metabolism of scoparone in human, mouse, rat, pig, dog, and rabbit liver microsomes in vitro and in humans in vivo. Oxidation of scoparone to isoscopoletin via 6-O-demethylation was the major metabolic pathway in liver microsomes from humans, mouse, rat, pig and dog, whereas 7-O-demethylation to scopoletin was the main reaction in rabbit. The scoparone oxidation rates in liver microsomes were 0.8 - 1.2 µmol/(min*g protein) in mouse, pig, and rabbit, 0.2 - 0.4 µmol/(min*g protein) in man and dog, and less than 0.1 µmol/(min*g) in rat. In liver microsomes of all species, isoscopoletin was oxidized to 3-[4-methoxy-ρ-(3, 6)-benzoquinone]-2-propenoate and esculetin, which was formed also in the oxidation of scopoletin. Human CYP2A13 exhibited the highest rate of isoscopoletin and scopoletin oxidation, followed by CYP1A1 and CYP1A2. Glucuronidation of isoscopoletin and scopoletin was catalyzed by the human UGT1A1, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, and UGT2B17. Dog was most similar to man in scoparone metabolism. Isoscopoletin glucuronide and sulfate conjugates were the major scoparone in vivo metabolites in humans, and they were completely excreted within 24 h in urine. Scoparone and its metabolites did not activate key nuclear receptors regulating CYP and UGT enzymes. These results outline comprehensively the metabolic pathways of scoparone in man and key preclinical animal species.

Molecular Docking-Based Design and Development of a Highly Selective Probe Substrate for UDP-glucuronosyltransferase 1A10.

Intestinal and hepatic glucuronidation by the UDP-glucuronosyltransferases (UGTs) greatly affect the bioavailability of phenolic compounds. UGT1A10 catalyzes glucuronidation reactions in the intestine, but not in the liver. Here, our aim was to develop selective, fluorescent substrates to easily elucidate UGT1A10 function. To this end, homology models were constructed and used to design new substrates, and subsequently, six novel C3-substituted (4-fluorophenyl, 4-hydroxyphenyl, 4-methoxyphenyl, 4-(dimethylamino)phenyl, 4-methylphenyl, or triazole) 7-hydroxycoumarin derivatives were synthesized from inexpensive starting materials. All tested compounds could be glucuronidated to nonfluorescent glucuronides by UGT1A10, four of them highly selectively by this enzyme. A new UGT1A10 mutant, 1A10-H210M, was prepared on the basis of the newly constructed model. Glucuronidation kinetics of the new compounds, in both wild-type and mutant UGT1A10 enzymes, revealed variable effects of the mutation. All six new C3-substituted 7-hydroxycoumarins were glucuronidated faster by human intestine than by liver microsomes, supporting the results obtained with recombinant UGTs. The most selective 4-(dimethylamino)phenyl and triazole C3-substituted 7-hydroxycoumarins could be very useful substrates in studying the function and expression of the human UGT1A10.

New glutathione conjugate of pyrrolizidine alkaloids produced by human cytosolic enzyme-dependent reactions in vitro.

RATIONALE: The toxic metabolites of pyrrolizidine alkaloids (PAs) are initially formed by cytochrome P450-mediated oxidation reactions and primarily eliminated as glutathione (GSH) conjugates. Although the reaction between the reactive metabolites and GSH can occur spontaneously, the role of the cytosolic enzymes in the process has not been studied. METHODS: The toxic metabolites of selected PAs (retrorsine, monocrotaline, senecionine, lasiocarpine, heliotrine or senkirkine) were generated by incubating them in 100 mM phosphate buffer (pH 7.4) containing liver microsomes of human, pig, rat or sheep, NADPH and reduced GSH in the absence or presence of human, pig, rat or sheep liver cytosolic fraction. The supernatants were analyzed using liquid chromatography connected to Finnigan LTQ ion-trap, Agilent QTOF or Thermo Scientific Q Exactive Focus quadrupole-orbitrap mass spectrometers. RESULTS: Retrorsine, senecionine and lasiocarpine yielded three GSH conjugates producing [M - H]- ions at m/z 439 (7-GSH-DHP (CHO)), m/z 441 (7-GSH-DHP (OH)) and m/z 730 (7,9-diGSH-DHP) in the presence of human liver cytosolic fraction. 7-GSH-DHP (CHO) was a novel metabolite. Monocrotaline, heliotrine and senkirkine did not produce this novel 7-GSH-DHP (CHO) conjugate. 7-GSH-DHP (CHO) disappeared when incubated with hydroxylamine, and a new oxime derivative was formed. This metabolite was formed only by the human liver cytosolic enzymes but not in the presence of rat or sheep liver cytosolic fractions under otherwise identical reaction conditions. CONCLUSIONS: 7-GSH-DHP (CHO) has not been reported before, and thus it was considered as a novel metabolite of PAs. This may clarify the mechanisms involved in PA detoxification and widely observed but less understood species differences in response to PA exposure.