Analysis of 4-bromo-3-fluorobenzaldehyde and separation of its regioisomers by one-dimensional and two-dimensional gas chromatography.

A starting material, 4-bromo-3-fluorobenzaldehyde, was used for active drug substance (API) AMG 369 production. The presence of the regioisomer impurities in the starting material 4-bromo-3-fluorobenzaldehyde presented significant challenges for the API synthetic route development due to the physical-chemical similarities of the impurities. These impurities significantly impact on the purity of the starting-material and final drug substance. Control of these impurities is important due to the potential genotoxicity of these impurities (p-GTI). Analytical development was carried out to develop GC methods with high resolving power and high sensitivity to quantify the regioisomers presented in starting material and therefore to control the purity of the starting material and the final drug substance. In the study, complete resolution of the ten regioisomers by 1D-GC and heart-cutting two-dimensional GC (2D-GC) was achieved. A sensitive GC/micro electron capture detection (µ-ECD) method with high resolving power and sensitivity to fully resolve all the ten regioisomers of 4-bromo-3-fluorobenzaldehyde was obtained by using a CHIRALDEX GC column (1D- GC). To facilitate the systematic GC method development, heart-cutting two-dimensional gas chromatography (2D-GC) using a Deans switch was exploited for the separation of the ten regioisomers. The resulting heart-cutting 2D-GC method successfully separated all the ten regioisomers with better sensitivity and resolution. Regioisomer impurities in the starting material were identified and quantified by these GC methods. The sensitivity for the methods is in the range of 0.004ng to 0.02ng for the regioisomers. Linearity for the methods is: R(2)=0.999 to 1.000. The methods were suitable for control of the regioisomer impurities, p-GTIs, in the starting material and final drug substance.

Mobile physician reporting of clinically significant events-a novel way to improve handoff communication and supervision of resident on call activities.

OBJECTIVES: Reporting of clinically significant events represents an important mechanism by which patient safety problems may be identified and corrected. However, time pressure and cumbersome report entry procedures have discouraged the full participation of physicians. To improve the process, our internal medicine training program developed an easy-to-use mobile platform that combines the reporting process with patient sign-out. METHODS: Between August 25, 2011, and January 25, 2012, our trainees entered clinically significant events into i-touch/i-phone/i-pad based devices functioning in wireless-synchrony with our desktop application. Events were collected into daily reports that were sent from the handoff system to program leaders and attending physicians to plan for rounds and to correct safety problems. RESULTS: Using the mobile module, residents entered 31 reportable events per month versus the 12 events per month that were reported via desktop during a previous 6-month study period. CONCLUSIONS: Advances in information technology now permit clinically significant events that take place during "off hours" to be identified and reported (via handoff) to next providers and to supervisors via collated reports. This information permits hospital leaders to correct safety issues quickly and effectively, while attending physicians are able to use information gleaned from the reports to optimize rounding plans and to provide additional oversight of trainee on call patient management decisions.

Evaluation of coronary artery stent patency by using 64-slice multi-detector computed tomography and conventional coronary angiography: a comparison with intravascular ultrasonography.

BACKGROUND: Most studies have investigated the diagnostic accuracy of 64-slice multi-detector computed tomography (MDCT) to detect coronary artery stent patency by using conventional coronary angiography (CCA) as the reference standard. In this study, we compared the diagnostic accuracy of MDCT and CCA by using intravascular ultrasonography (IVUS) as the reference standard. METHODS: Forty-six patients with previously implanted coronary artery stents (n=87) underwent MDCT followed by CCA and IVUS within 24h. Sensitivities, specificities, positive predictive values (PPV) and negative predictive values (NPV) of MDCT and CCA for detecting or excluding in-stent diameter restenosis (ISDR) by using in-stent area restenosis (ISAR) and minimal luminal area (MLA) ≤4.0 mm(2) of IVUS as the reference standard were determined. RESULTS: Eight stents (9%) were judged non-evaluable using MDCT for the detection of ISDR. ISDR was detected in 28% (22/79) of the evaluable stents using CCA. When ISAR was detected using IVUS, the sensitivity, specificity, PPV, and NPV for ISDR detection by using MDCT were 71%, 96%, 91% and 86%, and the corresponding values for CCA were 64%, 96%, 90% and 83%. When MLA ≤4.0mm(2) was detected using IVUS, the sensitivity, specificity, PPV, and NPV for ISDR detection by using MDCT were 87%, 96%, 91% and 95%, and for CCA were 78%, 96%, 90% and 92%. CONCLUSIONS: When ISAR with MLA ≤4.0 mm(2)was detected on IVUS, CCA and MDCT had similar diagnostic accuracies for ISDR detection. High specificity and NPV make 64-slice MDCT a reliable non-invasive method for excluding ISDR.

Endothelial dysfunction in young patients with acute ST-elevation myocardial infarction.

Endothelial dysfunction may be particularly important in the pathogenesis of young patients with acute myocardial infarction (AMI), because they have different clinical characteristics compared with older patients. We investigated endothelial function in relation to AMI in this young age group. From January 2005 to March 2008, 29 of 31 consecutive patients with acute ST-elevation myocardial infarction (STEMI) who were <40 years old and received direct percutaneous coronary intervention (PCI) were enrolled in the study. We compared the coronary risk factors and flow-mediated vasodilation (FMD) in the brachial artery between the acute STEMI patients and 29 age- and gender-matched controls that did not have AMI. Baseline brachial artery diameter and responses to glyceryl trinitrate were similar between the two groups. In contrast, FMD was significantly lower in the young acute STEMI group than in the control (3.47 ± 4.08 vs. 7.45 ± 4.67%, p = 0.001) and correlated with the Thrombolysis in Myocardial Infarction (TIMI) risk score. The impaired FMD in the acute STEMI group was independent of smoking, hyperlipidemia, hypertension, nitrate use, or body mass index. In multiple logistic regression analysis, only FMD and age, not traditional cardiovascular risk factors, were found to be significantly associated with acute STEMI (odds ratio = 0.75, 95% CI 0.63-0.90, p < 0.01). In conclusion, independent of conventional risk factors, severe endothelial dysfunction occurs in young acute STEMI patients and correlates with TIMI score. In addition to age, impaired FMD is the only significant factor associated with acute STEMI in this young population.

Six-year clinical follow-up after treatment of diffuse in-stent restenosis with cutting balloon angioplasty followed by intracoronary brachytherapy with liquid rhenium-188-filled balloon via transradial approach.

BACKGROUND: Long-term follow-up studies revealed a significant decline in the benefits of intracoronary radiation for in-stent restenosis. METHODS AND RESULTS: A total of 25 study and 25 contemporaneous control patients with diffuse in-stent restenosis who underwent cutting balloon angioplasty (CBA) transradially, followed by subsequent intracoronary irradiation with a liquid ß-emitter Rhenium-188 (¹88Re)-filled balloon were enrolled in the study. The mean clinical follow-up durations were 64.9 ± 13.0 and 66.3 ± 13.8 months for the irradiated and control patients, respectively. Six-month angiographic restenosis was observed in 16% (4 of 25) of the patients in the irradiated group and 48% (12 of 25) of the patients in the control groups (P = 0.03). The 6-month major adverse cardiac events (MACE) rate was 12% and 44%, respectively (P = 0.025). The 3-year follow-up angiography was performed in 16 of 21 (76%) irradiated patients and in 4 of 13 (31%) control patients who had no significant restenosis at the 6-month angiographic follow-up. Restenosis occurred in 1 of 16 (7%) irradiated patients and 2 of 4 (50%) control patients. Late target lesion revascularization was performed in 1 irradiated and 2 control patients. The MACE rate within 6 years was significantly reduced in the irradiated group (20% vs. 56%, P = 0.019). CONCLUSIONS: Brachytherapy using ¹88Re-filled balloon following CBA for diffuse in-stent restenotic native coronary arteries is effective in reducing target lesion restenosis and improving long-term outcomes.

Feasibility and safety of a transradial approach in intervention for chronic total occlusion of coronary arteries: a single-center experience.

BACKGROUND: The transradial approach for cardiac catheterization has become popular; however, its application in percutaneous coronary intervention (PCI) for chronic total occlusion (CTO) has rarely been reported. This study examines the feasibility and safety of this approach for performing PCI for CTO lesions. METHOD: We retrospectively evaluated 419 consecutive patients who underwent PCI for CTO lesions between February 1998 and December 2003 in our hospital; a transradial artery approach was used in 400 patients and a transfemoral artery approach in 19. RESULTS: The baseline clinical characteristics were similar in the 2 patient groups. The transradial group had more de novo lesions (76% vs 47.37%, p = 0.012), fewer in-stent restenotic lesions (11.75% vs 36.84%, p = 0.006) and smaller guiding catheters (p < 0.001) than the transfemoral group. There was no statistical difference in the procedure success rates (69.25% and 78.95%, p = 0.369) between the 2 groups. The incidence of major complications, including death, Q wave myocardial infarction, and emergency coronary artery bypass surgery, was similar in the 2 groups. CONCLUSIONS: The transradial approach for PCI can be a feasible choice for a CTO lesion. If this approach fails because of poor back up support from the guiding catheter, the transfemoral approach can be attempted with a larger guiding catheter.

Outcomes of interventions via a transradial approach for dysfunctional Brescia-Cimino fistulas.

The transradial artery approach to angioplasty has rarely been reported as a method for treating dysfunctional Brescia-Cimino fistulas. This study evaluated the feasibility, safety, and 1-year efficacy of this method for treating dysfunctional Brescia-Cimino fistulas. We retrospectively evaluated 154 consecutive procedures in 131 patients (age, 58.3 +/- 11.6 years; male, 48.1%) who underwent the transradial approach in dysfunctional Brescia-Cimino fistulas in the 1-year period after the procedure. The operator determined the use of a regular or a cutting balloon (two cases) in combination with urokinase injection (one case) or catheter thromboaspiration. Radial artery puncture was successful in all cases. Fifty-two cases (33.8%) had totally occluded fibrotic lesions. The overall anatomic success rate and clinical success rate were 61% (94/154) and 81.1% (125/154), respectively. In cases with a totally occluded fibrotic lesion, the clinical success rate was 46%. Successful intervention was associated with a significant reduction in the radial arterial systolic and diastolic pressures. There were no complications of symptomatic arterial embolization or pulmonary embolism, and one complication of venous rupture was successfully treated by compression. The primary patency rates based on intention-to-treat were 75.3% at 30 days and 39.0% at 1 year after the procedure. Excluding the cases with a totally occluded lesion, the clinical success rate was 99% (101/102) and the primary patency rates were 84.3% (86/102) and 52.0% (53/102) at 3 months and 1 year after the procedure, respectively. In conclusion, the transradial approach is a feasible, safe, and effective alternative for catheter intervention for dysfunctional Brescia-Cimino fistulas. Its success rate in cases with a totally occluded fibrotic lesion is unsatisfactory.

Prognostic predictors of sinus rhythm control by amiodarone and electrical cardioversion in patients undergoing percutaneous transluminal mitral valvuloplasty for rheumatic atrial fibrillation.

BACKGROUND: Conversion to sinus rhythm (SR) is rarely attempted in patients with rheumatic atrial fibrillation (AF) because the length of AF duration and the dilation of left atrium (LA) make maintenance of SR difficult. In this study, predictors of the successful maintenance of SR with amiodarone and electrical cardioversion in rheumatic AF patients receiving percutaneous transluminal mitral valvuloplasty (PTMV) were identified. METHODS AND RESULTS: This study included 23 consecutive patients undergoing PTMV for rheumatic AF (6 men, 53+/-11 years; AF duration 25 +/-24 months; LA diameter 44+/-6 mm; mitral valve area (MV) 1.1+/-0.2 cm(2)). Electrical cardioversion was required for the successful conversion to SR in all patients regardless of whether they had received amiodarone (400 mg/day) 2 months before PTMV (n=8) or 2 months after (n=15). After cardioversion, all patients received amiodarone 200 mg/day. With a follow-up period of 35+/-8 months, 14 patients (61%) remained in SR. A greater reduction in LA size (-4+/-3 mm vs 1+/-1 mm; p=0.004) and an greater increase in MV area (0.8+/-0.4 cm(2) vs 0.5+/-0.2 cm(2); p=0.01) by PTMV, not AF duration, were found to be the independent predictors for patients with successful maintenance of SR as compared with patients with recurrence of AF. CONCLUSION: In rheumatic AF patients receiving PTMV, the successful maintenance of SR with amiodarone and electrical cardioversion can be predicted by the degree to which LA size is reduced and MV area is increased.

Popliteal artery occlusion in a young baseball athlete.

Popliteal artery occlusion is a rare vascular complication in athletes and has not been previously documented in baseball players. A 21-year-old male baseball player presented with a 10-month history of progressive claudication because of repeated trauma-induced popliteal artery occlusion from frequently practicing stealing bases by sliding down onto his right leg. He was found to have a transient deficiency in both protein C and protein S. The patient underwent percutaneous transluminal recanalization angioplasty followed by anticoagulation therapy, with good results. This case illustrates the importance of awareness of this potential complication in baseball athletes, work-up for a hypercoagulable state and the feasibility of angioplasty therapy in the management of ischemic limbs after trauma.

Angiographic and clinical outcome in ST-segment elevation myocardial infarction patients receiving an adjunctive double bolus regimen of tirofiban for primary percutaneous coronary intervention.

BACKGROUND: Because of different dosages, the efficacy of adjunctive tirofiban therapy for primary percutaneous coronary intervention (PCI) is currently unclear. The hypothesis that a double bolus regimen of tirofiban will improve angiographic and clinical outcomes in patients with ST-segment elevation acute myocardial infarction (STEMI) undergoing PCI was tested in the present study. METHODS AND RESULTS: Primary PCI was performed in 217 STEMI patients: 80 received standard PCI (control group) and 137 received tirofiban (tirofiban group). Tirofiban was given as a bolus (10 mg/kg) in the emergency room and again upon arrival at the cardiac catheterization laboratory, followed by infusion of 0.15 mg . kg(-1) . min (-1) until the total dose reached 12.5 mg. The primary endpoint was emergency target vessel revascularization, recurrent myocardial infarction, or cardiovascular mortality at 30 days and 1 year. Baseline clinical and angiographic variables of the 2 groups were similar, as were angiographic results after PCI and bleeding complications at 30 days. The primary 30-day and 1-year endpoints were 5.1% and 11.7% in the tirofiban group, respectively, vs 10.0% (p = 0.171) and 18.8% (p = 0.151) in the control group. CONCLUSION: Although angiographic and clinical benefits were not demonstrated, the results suggest that research into an effective and uniform dosing regimen of adjunctive tirofiban therapy for PCI is warranted.

Membrane-associated STAT3 and PY-STAT3 in the cytoplasm.

Signal transduction from the plasma membrane to the nucleus by STAT proteins is widely represented as exclusively a soluble cytosolic process. Using cell-fractionation methods, we observed that approximately 5% of cytoplasmic STAT3 was constitutively associated with the purified early endosome (EE) fraction in human Hep3B liver cells. By 15-30 min after interleukin-6 (IL-6) treatment, up to two-thirds of cytoplasmic Tyr-phosphorylated STAT3 can be associated with the purified early endosome fraction (Rab-5-, EEA1-, transferrin receptor-, and clathrin-positive fraction). Electron microscopy, immunofluorescence, and detergent dissection approaches confirmed the association of STAT3 and PY-STAT3 with early endosomes. STAT3 was constitutively associated with clathrin heavy chain in membrane and in the 1- to 2-MDa cytosolic complexes. The membrane association was dynamic in that, within 15 min of treatment with the vicinal-thiol cross-linker phenylarsine oxide, there was a dramatic increase in bulk STAT3 association with sedimentable membranes. The functional contribution of PY-STAT3 association with the endocytic pathway was evaluated in transient transfection assays using IL-6-inducible STAT3-reporter-luciferase constructs and selective regulators of this pathway. STAT3-transcriptional activation was inhibited by expression constructs for dominant negative dynamin K44A, epsin 2a, amphiphysin A1, and clathrin light chain but enhanced by that for the active dynamin species MxA. Taken together, these studies emphasize the contribution of the endocytic pathway to productive IL-6/STAT3 signaling.

Atrial natriuretic peptide has dose-dependent, autonomically mediated effects on atrial refractoriness and repolarization in anesthetized dogs.

INTRODUCTION: Atrial natriuretic peptide (ANP) may alter electrophysiological properties of the heart and possibly have a role in arrhythmogenesis. However, previous studies have yielded conflicting results and have not fully considered whether ANP's cardiac electrophysiological effects are mediated via direct actions and/or indirectly via the autonomic nervous system. This study's aim was to establish whether ANP infused at pathophysiological and pharmacological doses has significant in vivo cardiac electrophysiological effects and to determine whether these effects are directly or autonomically mediated. METHODS AND RESULTS: Electrophysiologic and hemodynamic effects of ANP infusion (human ANP at 15-600 ng/kg per minute) were examined in chloralose-anesthetized dogs under conditions of varying autonomic blockade. In autonomically intact dogs (n = 12), low-dose ANP (15 ng/kg per minute) shortened atrial effective refractory period (ERP) (P < 0.001) and monophasic action potential duration (MAPD90) (P < 0.05) at 600, 500, and 400 msec atrial paced cycle lengths and reduced right atrial pressure (P < 0.05) but did not alter mean arterial pressure. After either combined vagal and beta-adrenergic blockade (vagotomy plus atropine plus propranolol, n = 7) or selective vagal blockade (n = 9), low-dose ANP no longer altered atrial ERP or MAPD90. Higher ANP doses (150 and 600 ng/kg per minute) decreased mean arterial and right atrial pressures (P < 0.001) but did not alter atrial ERP, MAPD90, or other electrophysiological parameters including atrial fibrillation threshold, ventricular ERP, and MAPD90. CONCLUSION: ANP has dose-dependent, autonomically mediated effects on atrial refractoriness and repolarization.

Different patterns of regulation of Tyr-phosphorylated STAT1 and STAT3 in human hepatoma Hep3B cells by the phosphatase inhibitor orthovanadate.

The cellular physiology of signal transducer and activator of transcription protein family (STAT) transcription factors includes activation by Tyr-phosphorylation (PY) in cytokine and growth factor receptor complexes at the level of plasma membrane rafts, subsequent cytoplasmic transit and nuclear import, and transcriptional regulation of target genes, followed by dephosphorylation and export back to the cytoplasm. The ubiquitous protein tyrosine phosphatase (PTP) called "T-cell protein tyrosine phosphatase" has been reported to mediate Tyr-dephosphorylation of both interferon-gamma (IFN-gamma)-induced PY-STAT1 and interkleukin-6 (IL-6)-induced PY-STAT3 in some cell lines. To test whether the same PTP regulated both PY-STAT1 and PY-STAT3 in human hepatocytes we used orthovanadate (VO(4); 0.01-1.0mM) as a PTP-inhibitory probe and evaluated the kinetics of PY-STAT3 and PY-STAT1 accumulation, nuclear trafficking, and dephosphorylation following cytokine (IL-6 or IFN-gamma) stimulation of Hep3B cells. As evaluated using DNA binding or Western blotting assays, in IL-6-treated hepatocytes VO(4) had a modest enhancing effect on peak levels of cytoplasmic and nuclear PY-STAT3 reached by 1h and on their subsequent decline. In contrast, in the same cells and at the same time, VO(4) caused a marked and continuing increase in cytoplasmic and nuclear levels of PY-STAT1 which, by 4h, were 5- to 10-fold higher than peak levels reached in VO(4)-free, IL-6-treated cells. Prolonged treatment of cells with VO(4) alone (for 4-8h) replicated this markedly selective enhancement of PY-STAT1 levels. Consistent with this selectivity, shorter term VO(4) treatment (1-2h) markedly increased PY-STAT1 levels in all cellular compartments of IFN-gamma-treated cells by >10-fold. The unexpected selectivity in the effects of VO(4) on PY-STAT1 compared to that on PY-STAT3 levels in Hep3B cells suggests that, at least in these hepatocytes, the regulation of PY-STAT1 and PY-STAT3 likely involves distinct protein tyrosine phosphatase mechanisms.

Association of the chaperone glucose-regulated protein 58 (GRP58/ER-60/ERp57) with Stat3 in cytosol and plasma membrane complexes.

Glucose-regulated protein 58 (GRP58/ER-60/ERp57), best known as a chaperone in the endoplasmic reticulum lumen, was previously identified by us as one of several accessory proteins in the S100 cytosol fraction of human hepatoma Hep3B cells that was differentially coshifted by anti-Stat3 antibody in an antibody-subtracted differential protein display assay. In the present study, the association between GRP58 and Stat3 in different cytoplasmic compartments was evaluated using cross-immunoprecipitation and cell-fractionation techniques. In the S100 cytosol fraction, three different anti-GRP58 polyclonal antibodies (pAb) cross-immunoprecipitated Stat3 (but not Stat1), and, conversely, anti-Stat3 pAb cross-immunoprecipitated GRP58. Both cytosolic Stat3 and GRP58 eluted during Superose-6 gel-filtration chromatography in complexes of size 200-400 kDa (statosome I), and anti-Stat3 pAb cross-immunoprecipitated GRp58 from these FPLC elution fractions. Using differential sedimentation and density equilibrium flotation methods, Stat3 and GRP58 were observed to be coassociated with cytoplasmic membranes enriched for the plasma membrane marker 5' nucleotidase but not with those containing the endoplasmic reticulum marker BiP/GRP78. The Stat3 and GRP58-containing plasma membrane fraction also contained Stat1, Stat5b, and gp130. Stat activation by orthovanadate caused the accumulation of PY-Stat3 in the GRP58-containing plasma membrane fraction. However, this PY-Stat3 was DNA-binding deficient. Likewise, excess exogenous recombinant human GRP58 prepared using a baculovirus expression system preferentially inhibited Stat3 DNA-binding activity in the S100 cytosol, suggesting that GRP58 may sequester activated Stat3. The new data confirm the association between GRP58 and Stat3 in cytosolic 200-400-kDa statosome I complexes and show that both GRP58 and Stat family members coassociate in the plasma membrane compartment. We suggest that the chaperone GRP58 may regulate signaling by sequestering inactive and activated Stat3.

Cytokine signaling: STATS in plasma membrane rafts.

STAT transcription factors signal from the plasma membrane to the nucleus in response to growth factors and cytokines. We have investigated whether plasma membrane "rafts" are involved in cytokine-activated STAT signaling. Cytokine-free human hepatoma Hep3B cells or cells treated with interleukin-6 (IL-6) or orthovanadate (a general activator of STATs) were fractionated, and plasma membrane raft fractions were obtained by equilibrium sedimentation or flotation through discontinuous sucrose gradients using either non-detergent or detergent-based (saponin or Triton X-100) methods. By Western blotting the plasma membrane raft fractions obtained using either non-detergent or detergent-based methods contained significant amounts of STAT1 and STAT3 (up to approximately 10% of the total cytoplasmic amount) as well as the integral raft proteins caveolin-1 and flotillin-1, the IL-6-receptor signal transducing chain gp130, the interferon-gamma receptor alpha chain (IFN-gammaRalpha), and the chaperone glucose-regulated protein 58 (GRP58/ER-60/ERp57). Upon activation of signaling by IL-6 or orthovanadate the respective Tyr-phosphorylated STAT species were now also observed in the membrane raft fraction but in a form deficient in DNA binding. The data show pre-association of STATs with plasma membrane rafts in flotation fractions, which also contained caveolin-1 and flotillin-1, and suggest that Tyr phosphorylation may not in itself be sufficient to cause the departure of PY-STATs from plasma membrane rafts. Methyl-beta-cyclodextrin, which sequesters cholesterol and disrupts plasma membrane rafts, markedly inhibited IL-6- and IFN-gamma-induced STAT signaling. Signaling through specialized raft microdomains may be a general mechanism operating at the level of the plasma membrane through which cytokines and growth factors activate STAT species (the "raft-STAT signaling hypothesis").